Thienoisoquinoline-phenylsulfonamides and their use as ER-NFκB inhibitors

ABSTRACT

This invention provides estrogen receptor modulators having the structure: 
                         
wherein R 1  to R 7  are as defined in the specification; or a pharmaceutically acceptable salt thereof.

RELATED APPLICATIONS

The present application claims benefit to U.S. Provisional ApplicationSer. No. 60/638,206 filed Dec. 21, 2004, which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

This invention relates to thienoisoquinoline-phenylsulfonamidecompounds, compositions containing them, their use as ER-NF_(k)Binhibitors, and methods of their preparation.

BACKGROUND OF THE INVENTION

The ability of ligands for the estrogen receptor to inhibit inflammatorygene expression (causing a reduction of cytokines, chemokines, adhesionmolecules and inflammatory enzymes) is believed to provide a means totreat the inflammatory component of diseases such as atherosclerosis,myocardial infarction (MI), congestive heart failure (CHF), inflammatorybowel disease and arthritis. Other potential therapeutic indications forthese type of molecules include type II diabetes (Cefalu, J WomensHealth & Gender-based Med. 2001, 10, 241 & Yuan et al., Science, 2001,293, 1673), osteoarthritis (Pelletier et al., Arthr. & Rheum.,2001,44:1237 and Felson et al. Curr Opinion Rheum, 1998, 10, 269) asthma(Chin-Chi Lin et. al., Immunol. Lett., 2000, 73, 57), Alzheiemer'sdisease (Roth, A. et. al., J. Neurosci. Res., 1999, 57, 399) andautoimmune diseases such as multiple sclerosis and rheumatoid arthritis.

A common component of these chronic inflammatory conditions is suspectedto be polymorphonuclear leukocyte and monocyte infiltration into thesite of damage through increased expression of cytokines and adhesionmolecules responsible for their recruitment. Overproduction of thecytokine interleukin (IL-6) has been associated with states of chronicinflammation (Bauer M. A., Herrmann F., Ann. Hematol., 1991, 62, 203).Synthesis of the IL-6 gene is induced by the transcription factornuclear factor κB (NF-κB). Interference at this step in the inflammatoryprocess can effectively regulate the uncontrolled proliferative processthat occurs in these chronic conditions.

In endothelial cells, 17β-estradiol (E2) inhibits IL-1β induced NF-κBreporter activity and IL-6 expression in an ER dependent fashion(Kurebayashi S. et. al., J. Steroid Biochem. Molec. Biol., 1997, 60,11). This has been said to correlate with anti-inflammatory action of E2in vivo as confirmed in different animal models of inflammation. Inmodels of atherosclerosis, E2 was shown to protect endothelial cellintegrity and function and to reduce leukocyte adhesion and intimalaccumulation (Adams, M. R. et al., Arterio., 1990, 1051, Sullivan, T. R.et al. J. Clin. Invst. 1995, 96, 2482, Nathan, L. et. al., Circ. Res.,1999, 85, 377). Similar effects of estrogen on the vascular wall havealso been demonstrated in animal models of myocardial infarction(Delyani, J. A. et al., J. Molec. Cell. Cardiol., 1996, 28, 1001) andcongestive heart failure. Clinically, estrogen replacement therapy (ERT)has been demonstrated to reduce the risk of mortality in patients withboth CHF (Reis et. al., J. Am. Coll. Cardio., 2000, 36, 529) and MI(Grodstein, F. et. al., Ann. Int. Med., 2000, 133, 933, Alexander et.al., J. Am. Coll. Cardio., 2001, 38, 1 and Grodstein F. et. al., Ann.Int. Med, 2001, 135,1). In ERT, clinical studies demonstrated aninfluence of E2 on the decrease in the production of β-amyloid 1-42(Aβ42), a peptide central for the formation of senile plaques inAlzheimer's disease (Schonknecht, P. et. al., Neurosci. Lett., 2001,307, 122).

17-β-estradiol, however, also strongly stimulates creatine kinaseexpression. Thus, in ERT some potential unwanted side effects, such asan increase risk of cardiovascular events in the first year of use, havebeen demonstrated (Hulley, S. et. al., J. Am. Med. Assoc., 1998, 280,605) as well as proliferative effects on uterine and breast tissue.

SUMMARY OF THE INVENTION

The instant invention relates to compounds that are able to serve asligands for the estrogen receptor. Preferred compounds arethienoisoquinoline-phenylsulfonamide compounds. In certain preferredembodiments, such compounds are of the formula:

where R₁ is hydrogen, alkyl, halogen, aryl, or heteroaryl; R₂, R₃, R₅,and R₆ are, independently, hydrogen, hydroxy, lower alkyl, alkoxy, orhalogen; R₄ is alkyl; and R₇ is hydrogen, —(C═O)R₁₆, —S(O)₂R₁₇,—S(O)₂N(R₁₈)(R₁₉), or D-glucuronidate, R₁₆ is alkyl, aralkyl or aryl,R₁₇ is alkyl, aryl, heteroaryl, cycloalkyl, alkenyl, cycloalkenyl, oralkynyl, R₁₈ and R₁₉ are, independently, hydrogen, alkyl, alkenyl,alkynyl, cycloalkyl, monofluoroalkyl, perfluoroalkyl, aryl, arylalkyl,cycloalkenyl, heteroaryl, heteroarylalkyl, hydroxy-(C₂-C₆)alkyl,alkoxyalkyl, alkylthioalkyl, carbonyl, acyl, alkoxycarbonyl, —C(O)NH₂,alkylaminocarbonyl, dialkylaminocarbonyl, alkylaminoalkyl, ordialkylaminoalkyl, or R₁₈ and R₁₉ are taken together with the nitrogenatom to which they are attached to form a saturated, unsaturated orpartially saturated C₄-C₆ carbon ring.

In certain embodiments, R₅ and R₆ are H.

In some embodiments, the compound is of the formula:

In some preferred embodiments, R₁ is methyl, ethyl, halogen, phenyl, orheteroaryl. In other preferred embodiments, R₁ is methyl, ethyl, bromo,phenyl, or thienyl. In certain compositions of the invention, R₂ is H orBr. In other compositions, R₃ is H or Br. R₂ and R₃ are each,independently, H or Br in yet other preferred compositions.

In certain preferred embodiments, R₄ is methyl or ethyl. In otherpreferred embodiments, R₇ is H or methyl.

In another aspect, the invention is drawn to pharmaceutical compositionsthat comprise one or more compounds of the invention and apharmaceutically acceptable carrier.

In yet other aspects, the invention concerns methods of treating orinhibiting chronic inflammatory disease in a mammal in need thereof,which comprise administering to said mammal an effective amount of acompound of the invention.

Such diseases include rheumatoid arthritis, spondyloarthropathies,osteoarthritis, psoriatic arthritis, juvenile arthritis, inflammatorybowel disease, Crohn's disease, ulcerative colitis, indeterminatecolitis, psoriasis, asthma and chronic obstructive pulmonary disease.

The invention is also directed to methods of treating or inhibitingstroke, ischemia, or reperfusion injury in a mammal in need thereof,which comprise administering to said mammal an effective amount of acompound of the invention. In other embodiments, the invention concernsmethods of lowering cholesterol, triglycerides, Lp(a), and LDL levels;inhibiting or treating hypercholesteremia, hyperlipidemia,cardiovascular disease, atherosclerosis, acute coronary syndrome,peripheral vascular disease, restenosis, or vasospasm in a mammal.

In yet other aspects, the compounds of the invention can be used fortreating or inhibiting Alzheimer's disease, cognitive decline, seniledementia, or type II diabetes in a mammal.

DETAILED DESCRIPTION OF THE INVENTION

Preferred compounds of the present invention are those that blockinterleukin-1β (IL-1β) induced nuclear factor κB (NF-κB) luciferasereporter activity or interleukin-6 (IL-6) expression in an ER dependentfashion in human endothelial cells. Compounds useful in the instantinvention show preferably little or no proliferative effects on uterineand breast tissue that is associated with estrogen in vivo. A lack ofestrogen side effects can be confirmed in vitro by the lack ofexpression of creatine kinase (CK), a classic estrogen responsive gene.The compounds described herein are expected to prove useful for thetreatment and prevention of chronic inflammatory diseases withoutstimulating uterine and breast cell proliferation as found with classicestrogens.

Compounds of the present invention includethienoisoquinoline-phenylsulfonamide compounds. In some aspects, thecompounds may be represented by the formula I as shown above.

In certain embodiments, OR₇ is in the position para to the sulfonylgroup. In some embodiments, OR₇ is meta to the sulfonyl group.

The instant invention is illustrated by the following compounds:

-   4-[(5-Methylthieno[3,2-c]isoquinolin4(5H)-yl)sulfonyl]phenol,-   4-[(5-Ethylthieno[3,2-c]isoquinolin-4(5H)-yl)sulfonyl]phenol,-   4-[(2-Bromo-5-methylthieno[3,2-c]isoquinolin-4(5H)-yl)sulfonyl]phenol,-   4-{[(5R)-2-bromo-5-methylthieno[3,2-c]isoquinolin-4-(5H)-yl]sulfonyl}phenol,-   4-{[(5S)-2-bromo-5-methylthieno[3,2-c]isoquinolin-4-(5H)-yl]sulfonyl}phenol,-   4-[(2-Bromo-5-ethylthieno[3,2-c]isoquinolin-4(5H)-yl)sulfonyl]phenol,-   4-[(5-Ethyl-2-thien-3-ylthieno[3,2-c]isoquinolin-4(5H)-yl)sulfonyl]phenol,-   4-[(5-Ethyl-2-phenylthieno[3,2-c]isoquinolin-4(5H)-yl)sulfonyl]phenol,-   2-Bromo-4-[(2-bromo-5-methylthieno[3,2-c]isoquinolin-4(5H)-yl)sulfonyl]phenol,-   2,6-Dibromo-4-[(2-bromo-5-methylthieno[3,2-c]isoquinolin-4(5H)-yl)sulfonyl]phenol.    and-   4-[(2,5-Diethylthieno[3,2-c]isoquinolin-4(5H)-yl)sulfonyl]phenol.

DESCRIPTION OF CHEMICAL GROUPS

The term “alkyl”, employed alone, is defined herein as, unless otherwisestated a C₁-C₂₀ monovalent saturated hydrocarbon moiety, eg. either a(C₁-C₂₀) straight chain or (C₃-C₂₀) branched-chain. Examples ofsaturated hydrocarbon alkyl moieties include, but are not limited to,chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl,tert-butyl, isobutyl, sec-butyl; higher homologs such as n-pentyl,n-hexyl, n-heptyl, n-octyl, and the like. It is preferred that straightchain alkyl moieties have 1-6 carbon atoms, and branched alkyl moietieshave 3-8 carbon atoms.

The term “alkenyl”, employed alone, is defined herein as, unlessotherwise stated, a C₂-C₂₀ monovalent hydrocarbon moiety containing atleast one double bond, e.g either a (C₂-C₂₀) straight chain or (C₃-C₂₀)branched-chain, Such hydrocarbon alkenyl moieties may be mono orpolyunsaturated, and may exist in the E or Z configurations. Thecompounds of this invention are meant to include all possible E and Zconfigurations. Examples of mono or polyunsaturated hydrocarbon alkenylmoieties include, but are not limited to, chemical groups such as vinyl,2-propenyl, isopropenyl, crotyl, 2-isopentenyl, butadienyl,2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), and higherhomologs, isomers, and the like. It is preferred that straight chainalkenyl moieties have 2-7 carbon atoms, and branched alkenyl moietieshave 3-8 carbon atoms.

The term “alkynyl”, employed alone, is defined herein as, unlessotherwise stated, a C₁-C₂₀ monovalent saturated hydrocarbon moiety,containing at least one triple bond, eg either a (C₂-C₂₀) straight chainor (C₃-C₂₀) branched-chain. Examples of alkynyl moieties include, butare not limited to, chemical groups such as ethynyl, 1-propynyl,1-(2-propynyl), 3-butynyl, and higher homologs, isomers, and the like.It is preferred that straight chain alkynyl moieties have 2-7 carbonatoms, and branched alkynyl moieties have 3-8 carbon atoms.

The term “alkylene” , employed alone or in combination with other terms,is defined herein as, unless otherwise stated, either a (C₁-C₂₀)straight chain or (C₂-C₂₀) branched-chain bivalent hydrocarbon moietyderived from an alkane; or a (C₂-C₂₀) straight chain or branched-chainbivalent hydrocarbon moiety derived from an alkene. Such hydrocarbonalkylene moieties may be fully saturated, or mono or polyunsaturated,and may exist in the E or Z configurations. The compounds of thisinvention are meant to include all possible E and Z configurations.Examples of saturated and unsaturated hydrocarbon alkylene moietiesinclude, but are not limited to bivalent chemical groups such as —CH₂—,—CH₂CH₂—, —CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂—, —CH═CH—, —CH═CHCH═CH—,vinylidene, and higher homologs, isomers, and the like. Preferredalkylene chains have 2-7 carbon atoms.

The term “cycloalkyl”, employed alone or in combination with otherterms, is defined herein as, unless otherwise stated, a monocyclic,bicyclic, tricyclic, fused, bridged, or spiro monovalent saturatedhydrocarbon moiety of 3-10 carbon atoms, wherein the carbon atoms arelocated inside or outside of the ring system. Any suitable ring positionof the cycloalkyl moiety may be covalently linked to the definedchemical structure. Examples of C₃-C₁₀ cycloalkyl moieties include, butare not limited to, chemical groups such as cyclopropyl,cyclopropylmethyl, cyclobutyl, cyclopentyl, cyclohexyl,cyclohexylmethyl, cyclohexylethyl, cycloheptyl, norbornyl, adamantyl,spiro[4.5]decanyl, and homologs, isomers, and the like.

The term “cycloalkenyl”, employed alone or in combination with otherterms, is defined herein as, unless otherwise stated, a monocyclic,bicyclic, tricyclic, fused, bridged, or spiro monovalent unsaturatedhydrocarbon moiety of 3-10 carbon atoms containing at least one doublebond, wherein the carbon atoms are located inside or outside of the ringsystem. Any suitable ring position of the cycloalkenyl moiety may becovalently linked to the defined chemical structure. Examples of C₃-C₁₀cycloalkenyl moieties include, but are not limited to, chemical groupssuch as cyclopropenyl, cyclopropenylmethyl cyclobutenyl, cyclopentenyl,cyclohexenyl, cyclohexenylmethyl, cyclohexenylethyl, cycloheptenyl,norbornenyl, and homologs, isomers, and the like.

The term “cycloalkylene”, employed alone, is defined herein as, unlessotherwise stated, a bivalent moiety of 3-10 carbon atoms derived from amonocyclic, bicyclic, tricyclic, fused, bridged, or spiro hydrocarbon.Such hydrocarbon cycloalkylene moieties may be fully saturated, or monoor polyunsaturated, and may exist in the E or Z configurations. Thecompounds of this invention are meant to include all possible E and Zconfigurations Any suitable ring position of the cycloalkylene moietymay be covalently linked to the defined chemical structure. Examples ofsaturated and unsaturated hydrocarbon cycloalkylene moieties include,but are not limited to, bivalent chemical groups such as cyclopropylene,cyclopentylene, cyclohexylene, cyclohexenylene,trans-decahydronaphthalenylene. spiro[3.3]heptenylene, and higherhomologs, isomers, and the like.

The terms “halo” or “halogen”, employed alone or in combination withother terms, is defined herein as, unless otherwise stated, a fluorine,chlorine, bromine, or iodine atom.

The term “monofluoroalkyl”, employed alone, is defined herein as, unlessotherwise stated, a C₁-C₁₀ monovalent saturated hydrocarbon moietycontaining only one fluorine atom, eg either a (C₁-C₁₀) straight chainor (C₃-C₁₀) branched-chain. Examples of monofluoroalkyl moietiesinclude, but are not limited to, chemical groups such as —CH₂F,—CH₂CH₂F, —CH(CH₃)CH₂CH₂F, and higher homologs, isomers, and the like.Preferred chain lengths are from 1-6 carbon atoms for straight chainsand from 3-8 carbon atoms for branched chains.

The term “monofluoroalkenyl”, employed alone, is defined herein as,unless otherwise stated, a C₂-C₁₀ monovalent unsaturated hydrocarbonmoiety containing only one fluorine atom and at least one double bond,eg either a (C₂-C₁₀) straight chain or (C₃-C₁₀) branched-chain. Examplesof monofluoroalkenyl moieties include, but are not limited to, chemicalgroups such as —CH═CH₂F, —CH₂CH═CH₂F, —CH═CHCH₂F, —C(CH₃)═CHF and higherhomologs, isomers, and the like. Preferred chain lengths are from 2-7carbon atoms for straight chains and from 3-8 carbon atoms for branchedchains.

The term “perfluoroalkyl”, employed alone or in combination with otherterms, is defined herein as, unless otherwise stated, a C₁-C₁₀monovalent saturated hydrocarbon moiety, containing two or more fluorineatoms eg, either a (C₁-C₁₀) straight chain or (C₃-C₁₀) branched-chain.Examples of perfluoroalkyl moieties include, but are not limited to,chemical groups such as trifluoromethyl, —CH₂CF₃, —CF₂CF₃, and—CH(CF₃)₂, and homologs, isomers, and the like. Preferred chain lengthsare from 1-7 carbon atoms for straight chains and from 3-8 carbon atomsfor branched chains.

The term “aryl”, employed alone or in combination with other terms, isdefined herein as, unless otherwise stated, an aromatic carbocyclicmoiety of up to 20 carbon atoms, (eg 6-20 carbon atoms), which may be asingle ring (monocyclic) or multiple rings (bicyclic, up to three rings)fused together or linked covalently. Any suitable ring position of thearyl moiety may be covalently linked to the defined chemical structure.Examples of aryl moieties include, but are not limited to, chemicalgroups such as phenyl, 1-naphthyl, 2-naphthyl, dihydronaphthyl,tetrahydronaphthyl, biphenyl, anthryl, phenanthryl, fluorenyl, indanyl,biphenylenyl, acenaphthenyl, acenaphthylenyl, and the like. It ispreferred that the aryl moiety contain 6-14 carbon atoms.

The term “arylalkyl”, employed alone or in combination with other terms,is defined herein as, unless otherwise stated, an aryl group, as hereinbefore defined, eg C₆-C₂₀ arylsuitably substituted on any open ringposition with an alkyl moiety wherein the alkyl chain is either a (C₁₋₇)straight or (C₂-C₇) branched-chain saturated hydrocarbon moiety.Examples of aryl(C₁-C₇)alkyl moieties include, but are not limited to,chemical groups such as benzyl, 1-phenylethyl, 2-phenylethyl,diphenylmethyl, 3-phenylpropyl, 2-phenylpropyl, fluorenylmethyl, andhomologs, isomers, and the like.

The term “heteroaryl”, employed alone or in combination with otherterms, is defined herein as, unless otherwise stated, an aromaticheterocyclic ring system, which may be a single ring (monocyclic) ormultiple rings (bicyclic, up to three rings) fused together or linkedcovalently and having for example five to twenty ring atoms. The ringsmay contain from one to four hetero atoms selected from nitrogen (N),oxygen (O), or sulfur (S), wherein the nitrogen or sulfur atom(s) areoptionally oxidized, or the nitrogen atom(s) are optionally substitutedor quarternized. Any suitable ring position of the heteroaryl moiety maybe covalently linked to the defined chemical structure. Examples ofheteroaryl moieties include, but are not limited to, heterocycles suchas furan, thiophene, pyrrole, N-methylpyrrole, pyrazole,N-methylpyrazole, imidazole, N-methylimidazole, oxazole, isoxazole,thiazole, isothiazole, 1H-tetrazole, 1-methyltetrazole,1,3,4-oxadiazole, 1H-1,2,4-triazole, 1-methyl-1,2,4-triazole,1,3,4-triazole, 1-methyl-1,3,4-triazole, pyridine, pyrimidine, pyrazine,pyridazine, benzoxazole, benzisoxazole, benzothiazole, benzofuran,benzothiophene, thianthrene, dibenzo[b,d]furan, dibenzo[b,d]thiophene,benzimidazole, N-methylbenzimidazole, indole, indazole, quinoline,isoquinoline, quinazoline, quinoxaline, purine, pteridine, 9H-carbazole,β-carboline, and the like.

The term “heteroarylalkyl”, employed alone or in combination with otherterms, is defined herein as, unless otherwise stated, a heteroarylgroup, as before defined, suitably substituted on any open ring positionwith a C₁-C₇ alkyl moiety, eg wherein the alkyl chain is either a(C₁-C₆) straight or (C₂-C₇) branched-chain saturated hydrocarbon moiety.Examples of heteroarylalkyl moieties include, but are not limited to,chemical groups such as furanylmethyl, thienylethyl, indolylmethyl, andthe like.

Heteroaryl chemical groups, as herein before defined, also includesaturated or partially saturated heterocyclic rings. Examples ofsaturated or partially saturated heteroaryl moieties include, but arenot limited to, chemical groups having 4-20 ring atoms such asazetidinyl, 1,4-dioxanyl, hexahydroazepinyl, piperazinyl, piperidinyl,pyrrolidinyl, morpholinyl, thiomorpholinyl, dihydrobenzimidazolyl,dihydrobenzofuranyl, dihydrobenzothienyl, dihydrobenzoxazolyl,dihydrofuranyl, dihydroimidazolyl, dihydroindolyl, dihydroisooxazolyl,dihydroisothiazolyl, dihydrooxadiazolyl, dihydrooxazolyl,dihydropyrrazinyl, dihydropyrazolyl, dihydropyridinyl,dihydropyrimidinyl, dihydropyrrolyl, dihydroquinolinyl,dihydrotetrazolyl, dihydrothiadiazolyl, dihydrothiazolyl,dihydrothienyl, dihydrotriazolyl, dihydroazetidinyl,dihydro-1,4-dioxanyl, tetrahydrofuranyl, tetrahydrothienyl,tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.

The term “acyl”, employed alone or in combination with other terms, isdefined herein as, unless otherwise stated, either an alkyl, arylalkyl,heteroarylalkyl, (C₂-C₁₀) straight chain, or (C₄-C₁₁) branched-chainmonovalent hydrocarbon moiety; wherein the carbon atom, covalentlylinked to the defined chemical structure, is oxidized to the carbonyloxidation state. Such hydrocarbon moieties may be mono orpolyunsaturated, and may exist in the E or Z configurations. Thecompounds of this invention are meant to include all possible E and Zconfigurations. Examples of C₂-C₁₁ acyl moieties include, but are notlimited to, chemical groups such as acetyl, propionyl, butyryl,3,3-dimethylbutyryl, trifluoroacetyl, pivaloyl, hexanoyl, hexenoyl,decanoyl, benzoyl, nicotinyl, isonicotinyl, and homologs, isomers, andthe like.

The term “hydroxyalkyl”, employed alone or in combination with otherterms, is defined herein as, unless otherwise stated, a (C₁-C₁₀)straight chain hydrocarbon, terminally substituted with a hydroxylgroup. Examples of hydroxyalkyl moieties include chemical groups such as—CH₂OH, —CH₂CH₂OH, —CH₂CH₂CH₂OH, and higher homologs.

The term “alkoxy”, employed alone or in combination with other terms isdefined herein as, unless otherwise stated, either a (C₁-C₁₀) straightchain or (C₃-C₁₀) branched-chain hydrocarbon covalently bonded to anoxygen atom. Examples of C₂-C₁₀ alkoxy moieties include, but are notlimited to, chemical groups such as methoxy, ethoxy, isopropoxy,sec-butoxy, tert-butoxy, decanoxy, and homologs, isomers, and the like.

The terms “aryloxy” or “heteroaryloxy”, employed alone or in combinationwith other terms, or unless otherwise stated, are aryl or heteroarylgroups, as herein before defined, which are further covalently bonded toan oxygen atom. Examples of aryloxy, or heteroaryloxy moieties include,but are not limited to, chemical groups such as C₆H₅O—, 4-pyridyl-O—,and homologs, isomers, and the like.

The term “carbonyl”, employed alone or in combination with other terms,is defined herein as, unless otherwise stated, a bivalent one-carbonmoiety further bonded to an oxygen atom with a double bond. An exampleis

The term “alkoxycarbonyl”, employed alone or in combination with otherterms, is defined herein as, unless otherwise stated, an alkoxy group,as herein before defined, which is further bonded to a carbonyl group toform an ester moiety. Examples of alkoxycarbonyl moieties include, butare not limited to, chemical groups such as methoxycarbonyl,ethoxycarbonyl, isopropoxycarbonyl, sec-butoxycarbonyl,tert-butoxycarbonyl, decanoxycarbonyl, and homologs, isomers, and thelike.

The term “alkylthio”, employed alone or in combination with other terms,is defined herein as, unless otherwise stated, an alkyl group aspreviously defined covalently bonded to a sulfur atom. Examples of(C₁-C₁₀)alkylthio moieties include, but are not limited to, chemicalgroups such as methylthio, ethylthio, isopropylthio, sec-butylthio,tert-butylthio, decanylthio, and homologs, isomers, and the like. It ispreferred that straight chain alkylthio moieties have 1-6 carbon atoms,and branched alkylthio moieties have 3-8 carbon atoms.

The terms “arylthio” or “heteroarylthio”, employed alone or incombination with other terms, or unless otherwise stated, are aryl orheteroaryl groups, as herein before defined, which are furthercovalently bonded to a sulfur atom. Examples of arylthio orheteroarylthio moieties include, but are not limited to, chemical groupssuch as C₆H₅S—, 4-pyridyl-S—, and homologs, isomers, and the like.

The terms “alkoxyalkyl” or “alkylthioalkyl”, employed alone or incombination with other terms, are an alkoxy or alkylthio group, asherein before defined, which is further covalently bonded to anunsubstituted (C₁-C₁₀) straight chain or unsubstituted (C₂-C₁₀)branched-chain hydrocarbon. Examples of alkoxyalkyl or alkylthioalkylmoieties include, but are not limited to, chemical groups such as,methoxymethyl, methylthioethyl, ethylthioethyl, isopropylthiomethyl,sec-butylthioethyl, —CH₂CH(CH₃)OCH₂CH₃ and homologs, isomers, and thelike. It is preferred that straight chain alkoxyalkyl or alkylthioalkylmoieties have 1-6 carbon atoms, and branched alkoxyalkyl oralkylthioalkyl moieties have 3-8 carbon atoms.

The terms “aryloxyalkyl”, “heteroaryloxyalkyl”, “arylthioalkyl”, or“heteroarylthioalkyl”, employed alone or in combination with otherterms, or unless otherwise stated, are aryloxy, heteroaryloxy, arylthio,or heteroarylthio groups, as herein before defined, which are furthercovalently bonded to an unsubstituted (C₁-C₁₀) straight chain orunsubstituted (C₂-C₁₀) branched-chain hydrocarbon. Examples ofaryloxyalkyl, heteroaryloxyalkyl, arylthioalkyl, or heteroarylthioalkylmoieties include, but are not limited to, chemical groups such asC₆H₅OCH₂—, C₆H₅OCH(CH₃)—, 4-pyridyl-O—CH₂CH₂—, C₆H₅SCH₂—, C₆H₅SCH(CH₃)—,4-pyridyl-S—CH₂CH₂—, and homologs, isomers, and the like. It ispreferred that straight chain aryloxyalkyl, heteroaryloxyalkyl,arylthioalkyl, or heteroarylthioalkyl moieties have 1-6 carbon atoms,and branched aryloxyalkyl, heteroaryloxyalkyl, arylthioalkyl, orheteroarylthioalkyl moieties have 3-8 carbon atoms.

The term “alkylamino”, employed alone or in combination with otherterms, or unless otherwise stated, is a moiety with one alkyl group,wherein the alkyl group is an unsubstituted (C₁-C₈) straight chainhereunto before defined alkyl group or an unsubstituted (C₃-C₈) hereuntobefore defined cycloalkyl group. Examples of alkylamino moietiesinclude, but are not limited to, chemical groups such as —NH(CH₃),—NH(CH₂CH₃), —NH-cyclopentyl, and homologs, and the like.

The term “dialkylamino”, employed alone or in combination with otherterms, or unless otherwise stated, is a moiety with two independentalkyl (including cycloalkyl) groups, wherein the alkyl groups areunsubstituted (C₁-C₆) straight chain hereunto before defined alkylgroups or unsubstituted (C₃-C₈) hereunto before defined cycloalkylgroups. Two groups may be linked to form an unsubstituted(C₂-C₆)-alkylene-group. Examples of dialkylamino moieties include, butare not limited to, chemical groups such as —N(CH₃)₂, —N(CH₂CH₃)₂,—NCH₃(CH₂CH₃),

and homologs, and the like.

The term “alkylaminoalkyl” employed alone or in combination with otherterms, or unless otherwise stated, is an alkylamino moiety, as hereinbefore defined, which is further covalently bonded to a straight chainalkyl group of 1-6 carbon atoms. Examples of alkylaminoalkyl moietiesinclude, but are not limited to, chemical groups such as —CH₂NH(CH₃),—CH₂CH₂NH(CH₂CH₃), —CH₂CH₂CH₂NH(CH₂CH₃), and homologs, and the like.

The term “dialkylaminoalkyl” employed alone or in combination with otherterms, or unless otherwise stated, is a dialkylamino moiety, as hereinbefore defined, which is further covalently bonded to a straight chainalkyl group of 1-6 carbon atoms. Examples of dialkylaminoalkyl moietiesinclude, but are not limited to, chemical groups such as —CH₂N(CH₃)₂,—CH₂CH₂N(CH₂CH₃)₂, —CH₂CH₂CH₂NCH₃(CH₂CH₃), and homologs, and the like.

The terms “alkylaminocarbonyl” or “dialkylaminocarbonyl”, employedalone, or unless otherwise stated, are alkylamino or dialkylaminomoieties, as herein before defined, which are further bonded to acarbonyl group. Examples of alkylaminocarbonyl or dialkylaminocarbonylmoieties include, but are not limited to, chemical groups such as—C(O)NH(CH₃), —C(O)N(CH₂CH₃)₂, —C(O)NCH₃(CH₂CH₃), and homologs, and thelike.

Each of the above terms (e.g., alkyl, aryl, heteroaryl) includesunsubstituted, monosubstituted, and polysubstituted forms of theindicated radical or moiety. Substituents for each type of moiety areprovided below.

Substituents for alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,alkylene, cycloalkylene, the alkyl portion of arylalkyl andheteroarylalkyl, saturated or partially saturated heterocyclic rings,and acyl or carbonyl moieties are, employed alone or in combination withother terms, selected from the group consisting of —R′, OR′, ═O, ═NR′,═N—OR′, —NR′R″, —SR′, halo, trifluoromethyl, trifluoromethoxy, —OC(O)R′,—CO₂R′, —C(O)NR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR″CO₂R′, —NR′C(O)NR′R″,—NH—C(NH₂)═NH, —NR′C(NH₂)═NH, —NH—C(NH₂)═NR′, —S(O)R′, —S(O)₂R′,—S(O)₂NR′R″, cyano, and nitro; wherein, R′ or R″ are each,independently, hydrogen, unsubstituted (C₁-C₆)alkyl, unsubstituted(C₃-C₇)cycloalkyl, aryl, aryl-(C₁-C₃)alkyl, aryloxy-(C₁-C₃)alkyl,arylthio-(C₁-C₃)alkyl, heteroaryl, heteroaryl-(C₁-C₃)alkyl,heteroaryloxy-(C₁-C₃)alkyl, or heteroarylthio-(C₁-C₃)alkyl groups; or ifoptionally taken together may be linked as an -alkylene-group eg 2-6carbon atoms, to form a ring.

The aryl or heteroaryl moieties, employed alone or in combination withother terms, may be optionally mono-, di- or tri-substituted withsubstituents selected from the group consisting of —R′, —OR′, —SR′,—C(O)R′, —CO₂R′, -alkoxyalkyl, alkoxyalkyloxy, cyano, halogen, nitro,trifluoromethyl, trifluoromethoxy, —NR′R″, alkylaminoalkyl,dialkylaminoalkyl, hydroxyalkyl, —S(O)R′, —S(O)₂R′, —SO₃R′, —S(O)₂NR′R″,—CO₂R′, —C(O)NR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR″CO₂R′, —NR′C(O)NR′R″,—NH—C(NH₂)═NH, —NR′C(NH₂)═NH, —NH—C(NH₂)═NR′, —S(O)R′, and —S(O)₂R′;wherein, R′ or R″ are each, independently, hydrogen, (C₁-C₆)alkyl,(C₃-C₇)cycloalkyl, aryl, aryl-(C₁-C₃)alkyl, aryloxy-(C₁-C₃)alkyl,arylthio-(C₁-C₃)alkyl, heteroaryl, heteroaryl-(C₁-C₃)alkyl,heteroaryloxy-(C₁-C₃)alkyl, or heteroarylthio-(C₁-C₃)alkyl groups; or ifoptionally taken together may be linked as an -alkylene-group eg 2-6carbon atoms, to form a ring.

A pro-drug is defined as a compound which is convertible by in vivoenzymatic or non-enzymatic metabolism (e.g. hydrolysis) to a compound ofthe invention wherein R₇ is a hydrogen atom.

The compounds of the present invention may contain an asymmetric atom,and some of the compounds may contain one or more asymmetric atoms orcenters, which may thus give rise to optical isomers (enantiomers) anddiastereomers. While the formulas herein are shown without respect tothe stereochemistry, the compounds of the present invention includessuch optical isomers (enantiomers) and diastereomers (geometricisomers); as well as the racemic and resolved, enantiomerically pure Rand S stereoisomers; as well as other mixtures of the R and Sstereoisomers and pharmaceutically acceptable salts thereof. Opticalisomers may be obtained in pure form by standard procedures known tothose skilled in the art, and include, but are not limited to,diasteromeric salt formation, kinetic resolution, and asymmetricsynthesis. It is also understood that this invention encompasses allpossible regioisomers, and mixtures thereof, which may be obtained inpure form by standard separation procedures known to those skilled inthe art, and include, but are not limited to, column chromatography,thin-layer chromatography, and high-performance liquid chromatography.

The compounds of the present invention may contain isotopes of atoms fordiagnostic, therapeutic, or metabolic purposes. Such isotopes may or maynot be radioactive.

The compounds of this invention include racemates, enantiomers,geometric isomers, or pro-drugs of the compounds shown herein.

Pharmaceutically acceptable salts of the compounds of compounds of theinstant invention with an acidic moiety can be formed from organic andinorganic bases. Suitable salts with bases are, for example, metalsalts, such as alkali metal or alkaline earth metal salts, for examplesodium, potassium, or magnesium salts; or salts with ammonia or anorganic amine, such as morpholine, thiomorpholine, piperidine,pyrrolidine, a mono-, di- or tri-lower alkylamine, where ‘lower’includes 1-6 carbon atoms, for example ethyl-tert-butyl-, diethyl-,diisopropyl-, triethyl-, tributyl- or dimethylpropylamine, or a mono-,di-, or trihydroxy lower alkylamine, for example mono-, di- ortriethanolamine. Internal salts may furthermore be formed. Similarly,when a compound of the present invention contains a basic moiety, saltscan be formed from organic and inorganic acids. For example salts can beformed from acetic, propionic, lactic, citric, tartaric, succinic,fumaric, maleic, malonic, mandelic, malic, phthalic, hydrochloric,hydrobromic, phosphoric, nitric, sulfuric, methanesulfonic,napthalenesulfonic, benzenesulfonic, toluenesulfonic, camphorsulfonic,and similarly known pharmaceutically acceptable acids.

As used in accordance with this invention, the term “providing,” withrespect to providing a compound or substance covered by this invention,means either directly administering such a compound or substance, oradministering a pro-drug, derivative, or analog which will form theeffective amount of the compound or substance within the body. Thisinvention also covers providing the compounds of this invention to treatthe disease states disclosed herein that the compounds are useful fortreating.

Solvates (e.g., hydrates) of the compounds of the present invention arealso within the scope of the present invention. Methods of solvation aregenerally known in the art. Accordingly, the compounds of the instantinvention can be in the free or hydrate form, and can be obtained bymethods exemplified by the following schemes below.

Also according to the present invention there is provided a method oftreating medical conditions which comprises administering to a human orother mammal an anti-inflammatory effective amount of a compound of thepresent invention. These conditions include atherosclerosis, myocardialinfarction, congestive heart failure, arthritis and inflammatory boweldisease in humans or other mammals.

It is understood that the effective dosage of the active compounds ofthis invention may vary depending upon the particular compound utilized,the mode of administration, the condition, and severity thereof, of thecondition being treated, as well as the various physical factors relatedto the individual being treated. For treating conditions such asatherosclerosis, myocardial infarction, congestive heart failure,arthritis and/or inflammatory bowel disease, generally satisfactoryresults may be obtained when the compounds of this invention areadministered to the individual in need at a daily dosage of from about0.1 mg to about 1 mg per kilogram of body weight, preferablyadministered in divided doses two to six times per day, or in asustained release form. For most large mammals, the total daily dosageis from about 3.5 mg to about 140 mg. In the case of a 70 kg humanadult, the total daily dose will generally be from about 7 mg to about70 mg and may be adjusted to provide the optimal therapeutic result.This regimen may be adjusted to provide the optimal therapeuticresponse.

The tablets, pills, capsules, and the like may also contain a bindersuch as gum tragacanth, acacia, corn starch or gelatin; excipients suchas dicalcium phosphate; a disintegrating agent such as corn starch,potato starch, alginic acid; a lubricant such as magnesium stearate; anda sweetening agent such as sucrose, lactose or saccharin. When a dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier such as a fatty oil. Various othermaterials may be present as coatings or to modify the physical form ofthe dosage unit. For instance, tablets may be coated with shellac, sugaror both. A syrup or elexir may contain, in addition to the activeingredients, sucrose as a sweetening agent, methyl and propyl parabensas preservatives, a dye and a flavoring such as cherry or orange flavor.

These active compounds may also be administered parenterally. Solutionsor suspensions of these active compounds can be prepared in watersuitably mixed with a surfactant such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols and mixtures thereof in oils. Under ordinary conditions ofstorage and use, there preparations contain a preservative to preventthe growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g. glycerol, propylene glycol and liquidpolyethylene glycol), suitable mixtures thereof, and vegetable oils.

Also according to the present invention there are provided processes forproducing the compounds of the present invention.

PROCESS OF THE INVENTION

Compounds of the invention can be readily prepared according to thefollowing reaction schemes or modifications thereof using readilyavailable starting materials, reagents and conventional syntheticprocedures. It is also possible to make use of variants of these processsteps, which in themselves are known to and well within the preparatoryskill of the medicinal chemist.

Some synthetic approaches to compounds of the present invention aredescribed in schemes 1-6. Starting with the readily available3-thiophene-carboxylic acid, diphenylphosphoryl azide and base in drytert-butanol (Scheme 1), heating to reflux can yield thetert-butylcarbonyl protected 3-aminothiophene 1 (J. Heterocycl. Chem.1968, 28, 417). Monobromination of the 2-position of the thiophene withN-bromosuccinimide in refluxing carbon tetrachloride can yield thethiophene 2 (J. Pharm. Sci. 1968, 5, 2003). Suziki coupling of thethiophene 2 with the boronic acid 3a,b (R═H, CH₃) under basic conditionsand then refluxing acid gives thienoisoquinolines 4a,b (4a ref. J.Heterocycl. Chem. 1989, 26, 865). Reduction with a hydride yields theamine 6, which is sulfonylated with a sulfonyl chloride and base in ahalogenated solvent to yield the sulfonamide 7. Demethylation of themethyl ether yields compound 8.

Alternatively, thienoisoquinoline 4a can be oxidized to the N-oxide 5with metachloro-peroxybenzoic acid in a halogenated solvent at roomtemperature. Reaction of the N-oxide 5 with a Grignard reagent, such asethyl magnesium bromide in ether at room temperature can yield the ethylsubstituted (R=ethyl) thienoisoquinoline 4c. Hydride reduction to theintermediate amine 6, followed by reaction with an aryl sulfonylchloride with base can be used to produce compound 7. Demethylation ofthe methyl ether can produce certain compounds of the present invention.

Halogenated thieno-isoquinolines can be synthesized from 4-alkoxyphenylsulfonamide 7 by reaction with N-bromosuccinimide in aceticacid-chloroform at −10° C. to room temperature to yield themono-bromothienoisoquinoline 9 (Scheme 3). Dealkylation of the alkylphenol with boron tribromide, cyclohexene, sodium bicarbonate in ahalogenated solvent at −78° C. to room temperature yields bromosubstituted thieno-isoquinoline sulfonamide 10.

Halogenated thienoisoquinolines can alternatively be synthesized from4-hydroxyphenyl sulfonamide 8 by reaction with N-bromosuccinimide inacetic acid-chloroform at room temperature to yield the target compound10 (Scheme 4), along with a dibromo compound and a tribromo compound.

The bromo-substituted thienoisoquinoline 9 can be reacted with anarylboronic acid or a heteroarylboronic acid in a Suzuki coupling with apalladium catalyst and base in an aqueous or non-aqueous solvent toyield an aryl or heteroaryl substituted thienoisoquinoline product 11(Scheme 5). Demethylation of the methyl ether with boron tribromide,cyclohexene and base in a halogenated solvent yields thethienoisoquinoline sulfonamide 12.

The halogen substituted thieno-isoquinoline sulfonamide 9 reacts withn-butyllithium then dimethylformamide to yield the carboxaldehyde 13(Scheme 6). Wittig reaction of the aldehyde yields the correspondingalkene 14. Demethylation of the methyl ether with boron tribromide,base, cyclohexene in a halogenated solvent yields the alkene substitutedthienoisoquinoline sulfonamide 15. The Wittig product 14 can also betreated with hydrogen and a palladium catalyst to yield, after work-up,the saturated compound 16. The demethylation of the methyl ether asmentioned previously yields the alkyl substituted thienoisoquinolinesulfonamide 17.

The following non-limiting examples further illustrate this invention.

EXPERIMENTAL

Commercially available reagents and solvents were used directly asreceived except for N-bromosuccinimide which was recrystallized fromwater. All procedures employing air- and/or moisture-sensitive reagentswere conducted under an inert atmosphere in flame-dried glassware whereappropriate. ¹H NMR spectra were recorded in DMSO-d₆ on a Varian Inovaspectrometer at 500 MHz, unless otherwise indicated.

EXAMPLE 1 Thieno[3,2-c]isoquinoline (Compound 4a)

Step 1: tert-Butyl thien-3-ylcarbamate (Compound 1)

3-Thiophenecarboxylic acid (31 g, 0.24 mol), Diphenylphosphoryl azide(57 mL, 0.26 mol) and triethylamine (36 mL, 0.26 mol) were combined indry tert-butanol (450 mL) and the resulting solution was heated toreflux. After 22 h the cooled solution was concentrated in vacuo toremove solvent. The residue was taken up in diethyl ether (1.5 L) andwashed with 5% aqueous citric acid, water and brine (1.5 L each), dried(Na₂SO₄), and concentrated in vacuo to give the crude product as a tackytan solid (51 g). Recrystallization from hot ethyl acetate-hexane gavethe title compound (33 g, 69%, m.p. 139-41° C.) as shiny off-whiteneedles.

¹H NMR δ 1.45 (s, 9 H, (CH₃)₃), 6.97 (m, 1 H, ArH), 7.16 (broad s, 1 H,ArH), 7.37 (m, 1 H, ArH), 9.60 (broad s, 1 H, NH). MS (ESI) m/z 198[M−H]⁻ Analysis calc. for C₁₈H₁₃FO₃S: C, 54.25; H, 6.58; N, 7.03. Found:C, 54.26; H, 6.48; N, 6.99.

Step 2: tert-Butyl 2-bromothien-3-ylcarbamate (Compound 2)

Tert-butyl thien-3-ylcarbamate (37 g, 0.19 mol) and N-bromosuccinimide(33 g, 0.19 mol) were combined in carbon tetrachloride (900 mL) andheated at reflux for 3 h. At this time, the mixture was cooled to 23° C.and filtered. The filtrate was washed with water (5×900 mL) to removesuccinimide, dried (Na₂SO₄), and concentrated in vacuo to give the titlecompound (50 g, 96%, m.p. 68-70° C.) as a tan solid which was pureenough for further use.

¹H NMR δ 1.43 (s, 9 H, (CH₃)₃), 7.09 (d, J=6 Hz, 1 H, ArH), 7.51 (d, J=6Hz, 1 H, ArH), 8.74 (broad s, 1 H, NH). MS (ESI) m/z 276 [M−H]⁻ Analysiscalc. for C₉H₁₂BrNO₂S: C, 38.86: H. 4.35: N, 5.04. Found: C, 38.66: H.4.10: N, 4.99.

Step 3: Thieno[3,2-c]isoquinoline (Compound 4a)

Tert-butyl 2-bromothien-3-ylcarbamate (35 g, 0.13 mol),2-Formylphenylboronic acid (Cmpd IIIa, 25 g, 0.17 mol),[1,1′-Bis(diphenylphosphino)ferrocene]dichloropalladium [II] (5.3 g, 6.5mmol), and sodium bicarbonate (33 g, 0.39 mol) were combined in 40%aqueous dimethoxyethane (675 mL) and the mixture was heated to reflux.After 1 h the mixture was cooled to 23° C., treated slowly with 2 Naqueous hydrochloric acid (405 mL) and heated back to reflux for anadditional 2 h. At this time the mixture was cooled to 23° C., treatedslowly with 2.5 N aqueous sodium hydroxide (270 mL) and stirred for ca.15 min. The mixture was filtered through Celite and washed with ethylacetate (2.2 L). The organic layer was separated from the filtrate andwashed with water and brine (2.2 L each), dried (K₂CO₃), andconcentrated in vacuo to give the crude product (28 g) as a dark oil.The oil was taken up in methylene chloride (75 mL) and pre-adsorbed onsilica gel (75 g). Flash chromatography on silica gel (725 g) eluting5-, then 10-, and then 20% ethyl acetate-hexane gave the title compound(19 g, 83%, m.p. 58-61° C.) as a yellow-brown solid which was pureenough for further use.

¹H NMR δ 7.71-7.74 (overlapping m, 2H, ArH), 7.89 (m. 1H, ArH), 8.06 (d,J=5.3 Hz, 1H, ArH), 8.17 (m, 1H, ArH), 8.27 (d, J=8 Hz, 1H, ArH), 9.29(s, 1H, ArH). MS (ESI) m/z 186 [M+H]⁺ Analysis calc. for C₁₁H₇NS: C,71.32; H, 3.81; N, 7.56. Found: C, 71.33; H, 3.79; N, 7.23.

EXAMPLE 2 5-Methylthieno[3,2-c]isoquinoline (Compound 4b)

Starting from 2-Acetylphenylboronic acid (15 g, 91 mmol) in place of2-formylphenylboronic acid in Step 3, the title compound (12 g, 86%,m.p. 74-76° C.) was synthesized in essentially the same manner asdescribed in Steps 1-3 for Thieno[3,2-c]isoquinoline.

¹H NMR δ 2.95 (s, 3H, CH₃), 7.62 (m, 1H, ArH), 7.71 (m, 1H, ArH), 7.86(m, 1H, ArH), 7.98 (m, 1H, ArH), 8.13 (d, J=8 Hz, 1H, ArH), 8.32 (d. J=8Hz. 1H, ArH). MS (ESI) m/z 200 [M+H]⁺ Analysis calc. for C₁₂H₉NS: C,72.33: H, 4.55; N. 7.03. Found: C. 72.13: H, 4.70: N, 7.01.

EXAMPLE 3 5-Ethylthieno[3,2-c]isoquinoline (Compound 4c)

Step 1: Thieno[3,2-c]isoquinoline-4-oxide (Compound 5)

Thieno[3,2-c]isoquinoline (13 g, 71 mmol) and 3-Chloroperoxybenzoic acid(57-86%, 21 g, 71 mmol) were combined in chloroform (300 mL) at 23° C.After 3 days the mixture was diluted with chloroform (750 mL), washedwith 10% aqueous sodium hydroxide (2×500 mL) and brine (500 mL), dried(MgSO₄), and concentrated in vacuo to give the crude product (13 g) as ayellow-brown solid. Recrystallization from hot ethanol (ca. 75 mL)provided the title compound (9.4 g, 66%, m.p. 190-95° C.) as dense brownneedles.

¹H NMR δ 7.67 (m, 1H, ArH), 7.71 (m, 1H, ArH), 7.84 (d, J=5.5 Hz, 1H,ArH), 8.01 (d, J=8 Hz, 1H, ArH), 8.10 (d, J=5.5 Hz, 1H, ArH), 8.12 (d,J=1H, ArH), 9.01 (s, 1H, ArH). MS (ESI) m/z 202 [M+H]⁺ Analysis calc.for C₁₁H₇NOS: C, 65.65; H, 3.51; N, 6.96. Found: C, 65.46; H, 3.46; N,6.89.

Step 2: 5-Ethylthieno[3,2-c]isoquinoline (Compound 4c)

A suspension of Thieno[3,2-c]isoquinoline 4-oxide (20 g, 99 mmol) in drytetrahydrofuran (ca. 400 mL) was treated dropwise at −78° C. with anethereal solution of ethylmagnesium bromide (3.0 M, 40 mL, 120 mmol)during 10 min. After 6 h the reaction mixture was quenched with 2 Naqueous hydrochloride acid (75 mL) and stirred for ca. 10 min. At thistime the solution was treated with 2.5 N aqueous sodium hydroxide (65mL). The resulting gelatinous residue was diluted with water (400 mL),filtered through Celite, and the filtrate was extracted with ethylacetate (3×500 mL). The combined extracts were washed with water andbrine (1.5 L each), dried (K₃CO₃) and concentrated in vacuo to give thecrude product (19 g) as an orange oil which was taken up in methylenechloride (ca. 60 mL) and pre-adsorbed on silica gel (60 g). Flashchromatography on silica gel (740 g) eluting 2.5-, then 5-, and then 10%ethyl acetate-hexane provided the title compound (17 g, 81%, m.p. 39-41°C.) as a clear, pale yellow oil which crystallized on standing.

¹H NMR δ 1.38 (m, 3H, CH₂CH₃), 3.36 (m, 2H, CH₂CH₃), 7.65 (d, J=5.4 Hz.1H, ArH), 7.70 (m, 1H, ArH), 7.86 (m, 1H, ArH), 7.99 (d, J=5.4 Hz, 1H,ArH), 8.14 (d, J=8 Hz, 1H, ArH), 8.37 (d, J=8 Hz, 1H, ArH). MS (EI) m/z213 [M]⁺ Analysis calc. for C₁₃H₁₁NS: C, 73.20; H, 5.20; N, 6.57. Found:C, 73.18; H, 5.14; N, 6.48.

EXAMPLE 4 4-[(5-Methylthieno[3,2-c]isoquinolin-4(5H)-yl)sulfonyl]phenol(Compound 8a)

Step 1: 5-Methyl-4,5-dihydrothieno[3,2-c]isoquinoline (Compound 6a)

5-Methylthieno[3,2-c]isoquinoline (12 g, 60 mmol) and sodium borohydride(9.0 g, 240 mmol) were combined in dry 1,4-dioxane (200 mL) at 23° C.Trifluoroacetic acid (9.2 mL, 120 mmol) was slowly added via syringewith gentle bubbling. After the addition was complete, the mixture washeated to reflux for 24 h. At this time the mixture was cooled to 23°C., quenched with de-gassed saturated aqueous sodium bicarbonate (450mL) and extracted under nitrogen with de-gassed methylene chloride(3×ca. 150 mL). The combined extracts were washed under nitrogen withde-gassed brine (ca. 450 mL), dried (Na₂SO₄) under nitrogen andconcentrated in vacuo to give the crude title compound (16 g) as anair-sensitive orange oil.

Step 2:4-[(4-Methoxyphenyl)sulfonyl]-5-methyl-4,5-dihydrothieno[3,2-c]isoquinoline(Compound 7a)

A solution of 4-Methoxybenzenesulfonyl chloride (16 g, 77 mmol),triethylamine (25 mL, 180 mmol), and 4-dimethylaminopyridine (0.20 g,1.6 mmol) in methylene chloride (100 mL) was de-gassed with nitrogen andtreated with a de-gassed methylene chloride (100 mL) solution of5-Methyl4,5-dihydrothieno[3,2-c]isoquinoline (16 g obtained above) at23° C. After 21 h the reaction mixture was quenched with 1 N aqueoussodium hydroxide (200 mL). The organic layer was separated and theaqueous phase was extracted with methylene chloride (2×200 mL). Thecombined extracts were washed with 1 N aqueous sodium hydroxide, 1 Naqueous hydrochloric acid, and brine (200 mL each), dried (K₂CO₃), andconcentrated in vacuo to give the crude product (21 g) as a dark oil.The oil was taken up in methylene chloride (ca. 80 mL) and pre-adsorbedon silica gel (80 g). Flash chromatography on silica gel (400 g) eluting25-, then 50-, and then 75% methylene chloride-hexane gave the product(16 g) as a yellow solid. Further elution with methylene chloride, then10-, and then 20% ethyl acetate-methylene chloride gave recovered5-methylthieno[3,2-c]isoquinoline (1.7 g, 14%) from the previous step.Recrystallization of the product from hot ethyl acetate-hexane providedthe title compound (15 g, 79% overall based on recovered startingmaterial from Step 1, m.p. 135-36° C.) as shiny yellow-orange prisms.

¹H NMR (400 MHz) δ 1.19 (d, J=7 Hz, 3H, CHCH₃), 3.63 (s, 3H, OCH₃), 5.43(m, 1H, CHCH₃), 6.64 (d, J=9 Hz, 2H, ArH), 7.01-7.13 (overlapping m, 5H,ArH), 7.22-7.26 (overlapping m, 2H, ArH), 7.57 (d, J=5.3 Hz, 1H, ArH).MS (ESI) m/z 372 [M+H]⁺ Analysis calc. for C₁₉H₁₇NO₃S₂: C, 61.43: H,4.61; N, 3.77. Found: C, 61.31: H, 4.56; N, 3.69.

Step 3: 4-[(5-Methylthieno[3,2-c]isoquinolin-4(5H)-yl)sulfonyl]phenol(Compound 8a)

A solution of4-[(4-methoxyphenyl)sulfonyl]-5-methyl-4,5-dihydrothieno[3,2-c]isoquinoline(0.45 g, 1.2 mmol) and tetrabutylammonium iodide (1.33 g, 3.6 mmol) indry methylene chloride (9 mL) was treated dropwise with a solution ofboron trichloride in methylene chloride (1.0 M, 3.6 mL, 3.6 mmol) at−78° C. during ca. 5 min. After a further 5 min., the solution waswarmed to 23° C. After 16 h the reaction solution was concentrated invacuo and the residue was taken up in ethyl acetate (25 mL). The organicphase was washed with saturated aqueous sodium bicarbonate (25 mL) andwater (10×25 mL), dried (MgSO₄), and concentrated in vacuo to give adark, oily solid (0.68 g) which was suspended in 20% ethylacetate-hexane and slurried onto a column of silica gel (10 g). Flashchromatography eluting 20-, and then 30% ethyl acetate-hexane gave theproduct (0.35 g) as a yellow solid. Recrystallization from hot ethylacetate-hexane gave the title compound (0.28 g, 65%, m.p. 163-65° C.) asshiny white needles.

¹H NMR (400 MHz) δ 1.18 (d, J=7 Hz, 3H, CHCH₃), 5.41 (m, 1H, CHCH₃),6.42 (d, J=9 Hz, 2H, ArH), 7.00-7.14 (overlapping m, 5H, ArH), 7.22-7.24(overlapping m, 2H, ArH), 7.56 (d, J=5 Hz, 1H, ArH), 10.3 (broad s, 1H,ArOH). MS (ESI) m/z 356 [M−H]⁻ Analysis calc. for C₁₈H₁₅NO₃S₂: C, 60.48;H, 4.23; N, 3.92. Found: C, 60.45; H, 4.11; N, 3.75.

EXAMPLE 5 4-[(5-Ethylthieno[3,2-c]isoquinolin-4(5H)-yl)sulfonyl]phenol(Compound 8b)

Starting from 5-ethylthieno[3,2-c]isoquinoline (12.8 g, 60 mmol) inplace of 5-methylthieno[3,2-c]isoquinoline in Step 1, the title compound(23 mg white solid, 24%, m.p. 169-71° C.) was synthesized in essentiallythe same manner as described in Example 1, Steps 1-3, for4-[(5-Methylthieno[3,2-c]isoquinolin-4(H)-yl)sulfonyl]phenol.

¹H NMR δ 0.89 (m, 3H, CH₂CH₃), 1.41 (m, 2H, CH₂CH₃), 5.10 (m, 1H,CHCH₂), 6.42 (d, J=9 Hz, 2H, ArH), 7.00-7.04 (overlapping m, 3H, ArH),7.08-7.14 (overlapping m, 2H, ArH), 7.21 (d, J=8 Hz, 1H, ArH), 7.24 (d,J=5 Hz, 1H, ArH), 7.55 (d, J=5 Hz, 1H, ArH), 10.2 (s, 1H, ArOH). HRMScalc. for C₁₉H₁₆NO₃S₂ [M−H]⁻: 370.05770. Found (ESI): 370.05778.

Method A

EXAMPLE 64-[(2-Bromo-5-methylthieno[3,2-c]isoquinolin-4(5H)-yl)sulfonyl]phenol(Compound 10a)

4-[(5-Methylthieno[3,2-c]isoquinolin4(H)-yl)sulfonyl]phenol (0.24 g,0.67 mmol) and N-bromosuccinimide (0.12 g, 0.67 mmol) were combined in1:1 acetic acid-chloroform (5 mL) at 23° C. After 24 h the resultingsolution was diluted with chloroform (45 mL) and washed with water, 25%aqueous potassium hydroxide, 1 N aqueous hydrogen chloride and brine (50mL each), dried (MgSO₄) and concentrated in vacuo to give the crudeproduct (0.28 g). Reverse phase HPLC (25×5 cm Primesphere 10 C₁₈ column,40% aqueous acetonitrile with 0.1% formic acid @ 100 mL/min) gave thetitle compound (0.12 g, 41%, m.p. 209-11° C.) as a white solid.

¹H NMR δ (d, J=7 Hz, 3H, CHCH₃), 5.37 (m, 1H, CHCH₃),6.47 (d, J=9 Hz,2H, ArH), 7.00 (d, J=7.5 Hz, 1H, ArH), 7.07-7.11 (overlapping m, 3H,ArH), 7.14 (m, 1H, ArH), 7.22 (d, J=7.5 Hz, 1H, ArH), 7.34 (s, 1H, ArH),10.3 (s, 1H, ArOH). HRMS calc. for C₁₈H₁₃BrNO₃S₂ [M−H]⁻ 433.95257. Found(ESI) 433.95249.

Method B

4-[(4-methoxyphenyl)sulfonyl]-5-methyl-4,5-dihydrothieno[3,2-c]isoquinolinewas separated via chiral HPLC utilizing a Chiralpak AS column and 9:1methanol:water as an eluent at 0.6 mL/min to yield the twoenantiomerically enriched compounds.

EXAMPLE 7 Step 1:(5R)-2-bromo-4-[(4-methoxyphenyl)sulfonyl]-5-methyl-4,5-dihydrothieno[3,2-c]isoquinoline(Compound 9a)

A solution of(5R)4-[(4-methoxyphenyl)sulfonyl]-5-methyl-4,5-dihydrothieno[3,2-c]isoquinoline(12.7 g, 34 mmol) in 1:1 acetic acid-chloroform (300 mL) was treatedwith N-bromosuccinimide (6.1 g, 34 mmol) at −10° C. After ca. 5 min.,the cooling bath was removed and the mixture was warmed to 23° C. After18 h the resulting solution was poured into water (1.2 L) and extractedwith methylene chloride (4×400 mL). The organic solution was washed with25% aqueous potassium hydroxide and brine (1 L each), dried (K₂CO₃), andconcentrated in vacuo to give the crude product (15.5 g) as a greensolid which was loaded onto a column of silica gel (100 g) in warm 25%methylene chloride-hexane. Flash chromatography eluting 25-, then 50-,and then 75% methylene chloride-hexane gave the title compound (15.1 g,98%, m.p. 136-38° C.) as a white solid. [α]_(D)=+219.4. ¹H NMR (400 MHz)δ 1.21 (d, J=7 Hz, CHCH₃), 3.64 (s, 3H, OCH₃), 5.40 (m, 1H, CHCH₃), 6.69(d, J=9 Hz, 2H, ArH), 6.98 (d, J=7.5 Hz, 1H, ArH), 7.08 (m, 1H, ArH),7.14 (m, 1H, ArH), 7.16-7.23 (overlapping m, 3H, ArH), 7.36 (s, 1H,ArH). MS (ESI) m/z 450 [M+H]⁺ Analysis calc. for C₁₉H₁₆BrNO₃S₂: C,50.67: H, 3.58; N, 3.11. Found: C, 50.68; H, 3.37; N, 3.04

Step 2:4-{[(5R)-2-bromo-5-methylthieno[3,2-c]isoquinolin-4-(5H)-yl]sulfonyl}phenol(Compound 10a)

A solution of(5R)-2-bromo-4-[(4-methoxyphenyl)sulfonyl]-5-methyl-4,5-dihydrothieno[3,2-c]isoquinoline(2.0 g, 4.4 mmol) in cyclohexene (34 mL) was treated with sodiumbicarbonate (11 g, 130 mmol) and the mixture was cooled to −78° C. Asolution of boron tribromide in methylene chloride (1.0 M, 22 mL, 22mmol) was added dropwise during ca. 10 min. At this time the coolingbath was removed and the reaction mixture was warmed to 23° C. After 75min the mixture was poured into saturated aqueous sodium bicarbonate(200 mL) and extracted with ethyl acetate (100 mL). The extract waswashed with water and brine (100 mL each), dried (Na₂SO₄), andconcentrated in vacuo to give a yellow gum (3.3 g) which was taken up inmethylene chloride (ca. 5 mL) and pre-adsorbed on silica gel (5 g).Flash chromatography on silica (145 g) eluting methylene chloride, then1-, and then 2% ethyl acetate-methylene chloride gave the title compound(0.81 g, 43%, m.p. 209-11° C.) as a pale yellow solid. The analyticalsample (EtOAc-hexane) gave pale yellow plates. [α]_(D)=+222.4.

¹H NMR δ 1.20 (d, J=7 Hz, 3H, CHCH₃), 5.37 (m, 1H, CHCH₃),6.47 (d, J=9Hz, 2H, ArH), 7.00 (d, J=7.5 Hz, 1H, ArH), 7.07-7.11 (overlapping m, 3H,ArH), 7.14 (m, 1H, ArH), 7.22 (d, J=7.5 Hz, 1H, ArH), 7.34 (s, 1H, ArH),10.3 (s, 1H, ArOH). MS (ESI) m/z 434 [M−H]⁻ Analysis calc. forC₁₈H₁₄BrNO₃S₂: C, 49.55; H, 3.23; N, 3.21. Found: C, 49.45, H. 3.25: N,3.10.

EXAMPLE 84-{[(5S)-2-bromo-5-methylthieno[3,2-c]isoquinolin-4-(5H)-yl]sulfonyl}phenol

This compound was synthesized in the same manner as in Example 7.[α]_(D)=−245.

EXAMPLE 94-[(2-Bromo-5-ethylthieno[3,2-c]isoquinolin-4(5H)-yl)sulfonyl]phenol

Starting from4-[(4-Methoxyphenyl)sulfonyl]-5-ethyl-4,5-dihydrothieno[3,2-c]isoquinolinein place of4-[(4-Methoxyphenyl)sulfonyl]-5-methyl-4,5-dihydrothieno[3,2-c]isoquinoline(Step 1), the title compound (0.19 g tan solid, 20%, m.p. 183-85° C.)was synthesized in essentially the same manner as described in Example6, Method B, Steps 1-2, for4-[(2-Bromo-5-methylthieno[3,2-c]isoquinolin-4(5H)-yl)sulfonyl]phenol.

¹H NMR δ 0.89 (m, 3H, CH₂CH₃), 1.43 (m, 2H, CH₂CH₃),5.07 (m, 1H, CHCH₂),6.46 (d, J=9 Hz, 2H, ArH), 7.00 (d, J=7.5 Hz, 1H, ArH), 7.08-7.11(overlapping m, 3H, ArH), 7.14 (m, 1H, ArH), 7.21 (d, J=7 Hz, 1H, ArH),7.34 (s, 1H, ArH), 10.2 (broad s, 1H, ArOH). HRMS calc. forC₁₉H₁₇BrNO₃S₂ [M+H]⁺: 449.98278. Found (ESI): 449.98221.

EXAMPLE 104-[(5-Ethyl-2-thien-3-ylthieno[3,2-c]isoquinolin-4(5H)-yl)sulfonyl]phenol(Compound 12a) Step 1:5-Ethyl-4-[(4-methoxyphenyl)sulfonyl]-2-thien-3-yl-4,5-dihydrothieno[3,2-c]isoquinoline(Compound 11a)

2-Bromo-5-ethyl-4-[(4-methoxyphenyl)sulfonyl]-4,5-dihydrothieno[3,2-c]isoquinoline(1.02 g, 2.2 mmol), 3-thiopheneboronic acid (0.37 g, 2.9 mol), potassiumcarbonate (0.91 g, 6.6 mol) and[1.1′-bis(diphenylphosphino)ferrocene]dichloropalladium [II] (90 mg,0.11 mmol) were combined in 40% aqueous dimethoxyethane (11 mL) and themixture was heated to 75° C. After 3.5 h the mixture was cooled to 23°C., filtered through Celite and washed with ethyl acetate (125 mL). Thefiltrate was washed with 1 N aqueous sodium hydroxide and brine (125 mLeach), dried (K₂CO₃), and concentrated in vacuo to give the crudeproduct (0.91 g) as a tan solid which was taken up in methylene chloride(ca. 3 mL) and pre-adsorbed on silica gel (3 g). Flash chromatography onsilica gel (27 g) eluting 25-, and then 50% methylene chloride-hexanegave the title compound (0.73 g, 71%, m.p. 182-84° C.) as a white solidwhich was pure enough for further use.

¹H NMR δ 0.93 (m, 3H, CH₂CH₃), 1.46 (m, 2H, CH₂CH₃), 3.63 (s, 3H, OCH₃),5.13 (m, 1H, CHCH₂), 6.66 (d, J=9 Hz, 2H, ArH), 7.00 (d, J=8 Hz, 1H,ArH), 7.08-7.14 (overlapping m, 2H, ArH), 7.19 (d, J=9 Hz, 2H, ArH),7.22 (d, J=8 Hz, 1H, ArH), 7.53 (m, 1H, ArH), 7.56 (s, 1H, ArH), 7.69(m, 1H, ArH), 7.90 (m, 1H, ArH). HRMS calc. for C₂₄H₂₂NO₃S₃ [M+H]⁺:468.07564. Found (ESI): 468.07667.

Step 2:4-[(5-Ethyl-2-thien-3-ylthieno[3,2-c]isoquinolin-4(5H)-yl)sulfonyl]phenol(Compound 12a)

A suspension of5-ethyl-4-[(4-methoxyphenyl)sulfonyl]-2-thien-3-yl-4,5-dihydrothieno[3,2-c]isoquinoline(0.73 g, 1.6 mmol) in cyclohexene (11 mL) was treated with sodiumbicarbonate (4.0 g, 48 mmol) and the mixture was cooled to −78° C. Asolution of boron tribromide in methylene chloride (1.0 M, 6.4 mL, 6.4mmol) was added dropwise during ca. 10 min. At this time the coolingbath was removed and the reaction mixture was warmed to 23° C. After 4 hthe mixture was poured into saturated aqueous sodium bicarbonate (75 mL)and extracted with ethyl acetate (50 mL). The extract was washed withbrine (75 mL), dried (Na₂SO₄), and concentrated in vacuo to give a darkoil (2.1 g) which was taken up in methylene chloride (ca. 5 mL) andpre-adsorbed on silica gel (5 g). Flash chromatography on silica (50 g)eluting 1.25-, and then 2.5% ethyl acetate-benzene gave the product(0.26 g) as a yellow solid. Recrystallization from hot ethylacetate-hexane gave the title compound (0.15 g, 21%, m.p. 195-96° C.) asshiny tan needles.

¹H NMR δ 0.92 (m, 3H, CH₂CH₃), 1.46 (m, 2H, CH₂CH₃), 5.10 (m, 1H,CHCH₂), 6.44 (d, J=9 Hz, 2H, ArH), 7.01 (m, 1H, ArH), 7.08 (d, J=9 Hz,2H, ArH), 7.10-7.14 (overlapping m, 2H, ArH), 7.21 (m, 1H, ArH), 7.52(m, 1H, ArH), 7.55 (s, 1H, ArH), 7.68 (m, 1H, ArH), 7.89 (m, 1H, ArH),10.2 (s, 1H, ArOH). MS (ESI) m/z 452 [M−H]⁻ Analysis calc. forC₂₃H₁₉NO₃S₃: C, 60.90: H, 4.22; N, 3.09. Found: C, 60.81; H, 4.20; N,2.95.

EXAMPLE 114-[(5-Ethyl-2-phenylthieno[3,2-c]isoquinolin-4(5H)-yl)sulfonyl]phenol(Compound 12b)

Starting from2-bromo-5-ethyl-4-[(4-methoxyphenyl)sulfonyl]-4,5-dihydrothieno[3,2-c]isoquinoline(1.02 g, 2.2 mmol) and substituting phenylboronic acid (0.35 g, 2.9mmol) in place of 3-thiopheneboronic acid in Step 1, the title compound(0.38 g, 50%, m.p. ° C.) was synthesized in essentially the same manneras described in Example 8, Steps 1-2, for4-[(5-ethyl-2-thien-3-ylthieno[3,2-c]isoquinolin-4(H)-yl)sulfonyl]phenol.

¹H NMR δ 0.92 (m, 3H, CH₂CH₃), 1.49 (m, 2H, CH₂CH₃), 5.12 (m, 1H,CHCH₂), 6.45 (d, J=9 Hz, 2H, ArH), 7.06-7.16 (overlapping m, 5H, ArH),7.23 (m, 1H, ArH), 7.36 (m, 1H, ArH), 7.46 (m, 2H, ArH), 7.65 (s, 1H,ArH), 7.74 (d, J=8 Hz, 2H, ArH), 10.3 (s, 1H, ArOH). MS (ESI) m/z 446[M−HR]⁻ Analysis calc. for C₂₅H₂₁NO₃S₂: C, 67.09; H, 4.73; N, 3.13.Found: C, 67.09; H, 5.03; N, 3.08.

EXAMPLE 12 and EXAMPLE 132-Bromo-4-[(2-bromo-5-methylthieno[3,2-c]isoquinolin-4(5H)-yl)sulfonyl]phenoland2,6-Dibromo-4-[(2-bromo-5-methylthieno[3,2-c]isoquinolin-4(5H)-yl)sulfonyl]phenol

Starting from4-[(5-Methylthieno[3,2-c]isoquinolin4(H)-yl)sulfonyl]phenol (0.72 g, 2.0mmol) and N-bromosuccinimide (0.78 g, 4.4 mmol, 2.2 eq), the titlecompounds were synthesized in essentially the same manner as describedin Example 6 for4-[(2-Bromo-5-methylthieno[3,2-c]isoquinolin4(H)-yl)sulfonyl]phenol.

2-Bromo-4-[(2-bromo-5-methylthieno[3,2-c]isoquinolin-4(5H)-yl)sulfonyl]phenol

Yield: 0.23 g white solid, 22%. ¹H NMR δ 1.21 (d, J=7 Hz, 3H, CHCH₃),5.40 (m, 1H, CHCH₃), 6.61 (d, J=9 Hz, 1H, ArH), 7.00 (d, J=8 Hz, 1H,ArH), 7.02 (m, 1H, ArH), 7.10 (m, 1H, ArH), 7.16 (m, 1H, ArH), 7.25-7.26(overlapping s, d, 2H, ArH), 7.34 (s, 1H, ArH), 11.2 (broad s, 1H,ArOH). MS (ESI) m/z 512 [M−H]⁻ Analysis calc. for C₁₈H₁₃Br₂NO₃S₂: C,41.96; H, 2.54; N, 2.72. Found: C, 41.98; H, 2.71; N, 2.51.

2,6-Dibromo-4-[(2-bromo-5-methylthieno[3,2-c]isoquinolin-4(5H)-yl)sulfonyl]phenol

Yield: 0.34 g white solid, 29% (based on conversion of startingmaterial). ¹H NMR δ 1.18 (d, J=7 Hz, 3H, CHCH₃), 5.32 (m, 1H, CHCH₃),7.01-7.03 (overlapping s, d, 3H, ArH), 7.10 (m, 1H, ArH), 7.17 (m, 1H,ArH), 7.25 (d, J=8 Hz, 1H, ArH), 7.29 (s, 1H, ArH). MS (ESI) m/z 590[M−H]⁻ Analysis calc. for C₁₈H₁₂Br₃NO₃S₂: C, 36.39; H, 2.04; N, 2.36.Found: C, 36.36; H, 2.31; N, 2.12.

EXAMPLE 144-[(2-Formyl-5-ethylthieno[3,2-c]isoquinolin-4(5H)-yl)sulfonyl]phenolStep 1:5-Ethyl-4-[(4-methoxyphenyl)sulfonyl]-4,5-dihydrothieno[3,2-c]isoquinoline-2-carbaldehyde(Compound 13)

A solution of2-bromo-4-[(4-methoxyphenyl)sulfonyl]-5-ethyl-4,5-dihydrothieno[3,2-c]isoquinoline(2.3 g, 5.0 mmol) in dry tetrahydrofuran (45 mL) was cooled to −78° C.and treated with a solution of butyllithium in hexanes (1.35 M, 3.7 mL,5.0 mmol) during ca. 2 min followed immediately by the fast addition ofdry dimethylformamide (1.9 mL, 25 mmol). After 30 min further at −78°C., the reaction was quenched with 10% aqueous ammonium chloride (100mL) and extracted with ethyl acetate (3×50 mL). The combined extractswere washed with brine (75 mL), dried (MgSO₄), and concentrated in vacuoto give the crude product as a light orange solid (2.0 g) which wastaken up in methylene chloride (ca. 8 ML) and pre-adsorbed on silica gel(8 g). Flash chromatography on silica gel (72 g) eluting 50-, then 75%methylene chloride-hexane, and then methylene chloride gave the titlecompound (1.7 g, 81%, m.p. 129-32° C.) as a pale yellow solid.

¹H NMR δ 0.92 (m, 3H, CH₂CH₃), 1.44 (m, 2H, CH₂CH₃), 3.63 (s, 3H, OCH₃),5.20 (m, 1H, CHCH₂), 6.66 (d, J=9 Hz, 2H, ArH), 7.14-7.20 (overlappingm, 3H, ArH), 7.23 (d, J=8 Hz, 1H, ArH), 7.26 (m, 1H, ArH), 7.31 (d, J=8Hz, 1H, ArH), 8.18 (s, 1H, ArH), 9.99 (s, 1H, CHO). MS (EI) m/z 413 [M]⁺Analysis calc. for C₂₁H₁₉NO₄S₂: C, 61.00: H, 4.63; N, 3.39. Found: C,60.61; H, 4.73; N, 3.27.

Step 2:5-Ethyl-4-[(4-methoxyphenyl)sulfonyl]-2-vinyl-4,5-dihydrothieno[3,2-c]isoquinoline(Compound 16)

A suspension of methyltriphenylphosphonium bromide (2.30 g, 6.44 mmol)in dry tetrahydrofuran (16 mL) was treated dropwise with a pentanesolution of t-butyllithium (1.0 M, 3.9 mL, 3.9 mmol) at 0° C. duringseveral min. After the addition was complete, the suspension was warmedto 23° C. and after a further 10 min, a solution of5-ethyl-4-[(4-methoxyphenyl)sulfonyl]-4,5-dihydrothieno[3,2-c]isoquinoline-2-carbaldehyde(1.33 g, 3.22 mmol) in dry tetrahydrofuran (16 mL+8 mL wash) was added.After a further 15 min, the reaction was quenched with water (160 mL)and extracted with ethyl acetate (160 mL). The extract was washed withbrine (160 mL), dried (Na₂SO₄), and concentrated in vacuo to give thecrude product as an orange solid (2.46 g). Flash chromatography onsilica gel (24 g) eluting 25-, and then 50% methylene chloride-hexanegave the title compound (1.20 g, 90%, m.p. 131-33° C.) as a pale yellowsolid.

¹H NMR δ 0.90 (m, 3H, CH₂CH₃), 1.43 (m, 2H, CH₂CH₃), 3.63 (s, 3H, OCH₃),5.11 (m, 1H, CHCH₂), 5.26 (d, J=11 Hz, 1H, CHH═), 5.61 (d, J=17 Hz, 1H,CHH═), 6.66 (d, J=9 Hz, 2H, ArH), 6.93-7.00 (overlapping m, 2H, ArCH═,ArH), 7.07-7.14 (overlapping m, 2H, ArH), 7.17 (d, J=9 Hz, 2H, ArH),7.21 (d, J=7 Hz, 1H, ArH), 7.29 (s, 1H, ArH). MS (EI) m/z 411 [M]⁺Analysis calc. for C₂₂H₂₁NO₃S₂: C, 64.21; H, 5.14; N, 3.40. Found: C.63.85: H. 5.25: N, 3.31.

Step 3:2,5-Diethyl-4-[(4-methoxyphenyl)sulfonyl]-4,5-dihydrothieno[3,2-c]isoquinoline(Compound 16)

A solution of5-ethyl-4-[(4-methoxyphenyl)sulfonyl]-2-vinyl4,5-dihydrothieno[3,2-c]isoquinoline(1.44 g, 3.50 mmol) in ethyl acetate (35 mL) was hydrogenated over 10%palladium-on-carbon (0.29 g) at 23° C. and 1 atm. After 1 h the catalystwas filtered (Celite), washed with ethyl acetate (35 mL), and thefiltrate was concentrated in vacuo to give the crude product (1.37 g) asa pale yellow solid. Recrystallization from boiling hexane provided thetitle compound (1.26 g, 87%, m.p. 105.0-105.5° C.) as shiny pale yellowneedles.

¹H NMR δ 0.89 (m, 3H, CH₂CH₃), 1.28 (m, 3H, CH₂CH₃), 1.41 (m, 2H,CH₂CH₃), 2.86 (m, 2H, CH₂CH₃), 3.62 (s, 3H, OCH₃), 5.07 (m, 1H, CHCH₂),6.65 (d, J=9 Hz, 2H, ArH), 6.92 (m, 1H, ArH), 7.01 (s, 1H, ArH),7.03-7.09 (overlapping m, 2H, ArH), 7.14 (d, J=9 Hz, 2H, ArH), 7.17 (m,1H, ArH). MS (ESI) m/z 414 [M+H]⁺ Analysis calc. for C₂₂H₂₃NO₃S₂: C,63.90; H, 5.61; N, 3.39. Found: C, 63.78; H, 5.52; N, 3.31.

Step 4: 4-[(2,5-Diethylthieno[3,2-c]isoquinolin-4(5H)-yl)sulfonyl]phenol(Compound 17)

Starting from2,5-diethyl-4-[(4-methoxyphenyl)sulfonyl]4,5-dihydrothieno[3,2-c]isoquinoline(1.26 g, 3.05 mmol) in place of2-bromo-4-[(4-methoxyphenyl)sulfonyl]-5-methyl4,5-dihydrothieno[3,2-c]isoquinoline,the title compound (0.87 g, 71%, m.p. 119-122° C.) was synthesized inessentially the same manner as described in Example 6, Method B, Step 2.

¹H NMR δ 0.89 (m, 3H, CH₂CH₃), 1.28 (m, 3H, CH₂CH₃), 1.41 (m, 2H,CH₂CH₃), 2.85 (m, 2H, CH₂CH₃), 5.05 (m, 1H, CHCH₂), 6.44 (d, J=9 Hz, 2H,ArH), 6.94 (m, 1H, ArH), 7.00 (s, 1H, ArH), 7.04 (d, J=9 Hz, 2H, ArH),7.06-7.08 (overlapping m, 2H, ArH), 7.17 (m, 1H, ArH), 10.2 (s, 1H,ArOH). MS (ESI) m/z 400 [M+H]⁺ Analysis calc. for C₂₁H₂₁NO₃S₂: C. 63.13;H. 5.30: N, 3.51. Found: C. 62.86: H. 5.48: N, 3.14.

EXAMPLE 15

In Vitro Methods

NFκB-Luciferase, IL-6 and Creatine Kinase Assays

Cells

T-175 flasks of 100% confluent HAECT-1 cells (immortalized human aorticendothelial cells) were washed with 8 ml of HBSS (HEPES buffered salinesolution) and infected for four hours with 6 ml of a 1:10 dilution ofAd5-wt-hER virus (an adenovirus transfection vector that mediates CMVpromoter driven expression of human ER) in phenol red free EndothelialCell Basal medium (Clonetics, San Diego Calif., Catalog # CC-3129)containing 0.25% bovine serum albumin (EBM-BSA). After four hours, cellswere washed with EBM-BSA and incubated overnight in the same medium.Following overnight incubation, cells were washed with EBM-BSA andinfected for 2 hours with 6 ml of a 1:10 dilution of Ad5-3×(NF B).Lucvirus (Adenovirus luciferase expression vector driven by 3 repeats ofthe MHC NF b site 5′ to the thymidine kinase promoter) in EBM-BSA. Aftertwo hours, cells were washed and incubated at 34° C. for 1 hour. Cellswere then washed, trypsinized, counted and resuspended in 95% FBS/5%dimethylsulfoxide at a concentration of 4×10⁶ cells/ml, frozen as 1 or 5ml aliquots in cryo-vials and stored at −150° C. Control (no ERinfection) cells were processed as above without Ad5-wt-hER virusinfection.

Assays

ER infected HAECT-1 cells or control cells were thawed, diluted 42× inwarm EBM-BSA, plated into 96-well plates at 0.1 ml/well and incubatedfor 4 h at 34° C. Test compounds were added to the cells as 2× stocks inEBM-BSA containing 2 ng/ml IL-1 (R&D Systems) and plates were returnedto the incubator (34° C.). After 15-20 h, 100 l aliquots of media wereremoved from the cells and assayed for IL-6 content using a BioSourcehuman IL-6 ELISA Kit. Cells were subsequently washed with 300 l ofDulbecco's phosphate buffered saline and lysed in 50 l of Cell CultureLysis Reagent (Promega). Luciferase was determined on a Wallac Victor²Luminometer (Gaithersburg, Md.) using 10 l of lysate and mixing with 100l of Promega Luciferase Assay reagent. Creatine kinase was determinedfrom the rate of increase in A₃₄₀ following addition of 100 l of CKassay reagent (Sigma, cat. No 47-10) to the remainder of the celllysate.

IL-6 Promoter Luciferase Assay

HAECT-1 cells (immortalized human aortic endothelial cells) in 96-wellplates were infected for two hours with 50 l/well of a 1:10 dilution ofAd5-wt-hER virus in EBM-BSA. After two hours, virus was removed, 100 lof EBM-BSA was added per well. Following overnight incubation, EBM-BSAwas removed and cells were infected for 2 hours with 50 l/well of a 1:10dilution of Ad5-IL6(1250 bp).Luc virus (Adenovirus luciferase expressionvector driven by a 1250 bp section of the human IL-6 promoter 5′ to thethymidine kinase promoter) in EBM-BSA. After two hours, virus wasremoved and plates were incubated at 34° C. for 1-2 h. Cells weresubsequently treated with test compounds/IL-1 and luciferase activitywas determined as above.

Data Analyses

For IC₅₀ and EC₅₀ calculations, mean IL-6, luciferase or CK valuesversus log10 of the compound concentration were fitted to a fourparameter logistic equation. The IC₅₀/EC₅₀ value, ‘Hill slope’, upperand lower limits of the curve were iteratively estimated.

Mice

Ovariectomized C57BL/6 mice (16-20 g) (Taconic) were separated intogroups of 8. After 5-7 days of recuperation, the mice were fed a chowdiet or an atherogenic diet (15.75% fat, 1.25% cholesterol and 0.5%sodium cholate) (Purina diet #21539). EE or test compound wasadministered once daily by gavage in a methylcellulose/tween vehicle(0.1 ml per mouse) for 5 weeks. At the end of the experimental period,the liver was collected and uterine wet weight was recorded.

RNA Analysis

Liver total RNA was prepared by using Trizol reagent (BRL). Estrogen andcompound regulation of NF-B target genes were verified by real timeRT-PCR using an ABI PRISM 7700 Sequence Detection System according tothe manufacturer's protocol (Applied Biosystems). The data was analyzedusing the Sequence Detector v1.7 software (Applied Biosystems) andnormalized to GAPDH using the Applied Biosystems primer set.

In vitro Results

Table 1 summarizes the activities of compounds contained herein in theHAECT-1 NF-κB, IL-6 and creatine kinase assays in Ad5-wt-ER infectedcells and is compared to activities of the same compounds in the HAECT-1NF-κB and creatine kinase assays in uninfected cells.

TABLE 1 Effects of 17-β-estradiol on NF-κB. IL-6 and CK expression inAd5-wt-ER infected HAECT-1 cells ER/NF-KB-luc ER/IL-6 ER/CK Example IC₅₀% IC₅₀ % EC₅₀ % # (nM) Eff* (nM) Eff (nM) Eff* E2 1 100 1.7 100 5.8 1004 66.8 172 100 42 5 21.3 174 N/O 6 28.2 162 N/O 7 1451 84 5822 161 N/O 819 92 8.9 164 N/O 9 7.7 194 47 33 10 11 24.6 132 15 63 12 1475 197 154335 13 N/O - activity not observed at doses tested. *Efficacy values arerelative to the maximal inhibition (NF-κB or IL-6 assay) or stimulation(CK assay) observed with E2

E2 inhibits NF-κB and IL-6 expression in Ad5-wt-ER infected HAECT-1cells with an IC₅₀ value around 1 nM and induces expression of creatinekinase in the same cells with similar potency (5.8 nM). In contrast,compounds of the present invention potently and efficaciously inhibitNF-κB and IL-6 expression in Ad5-wt-ER infected HAECT-1 cells showlittle or no CK expression (Table 1) in an ER-dependent manner. Theability of compounds of the present invention to inhibit NF-κB and IL-6expression without inducing CK activity (Table 1) is consistent with ananti-inflammatory activity in the absence of classic estrogenicactivity.

Evaluation of Test Compound Using an ERE-Reporter Test Procedure inMCF-7 Breast Cancer Cells

Stock solutions of test compounds (usually 0.1 M) are prepared in d thendiluted 10 to 100-fold with DMSO to make working solutions of 1 or 10mM. The DMSO stocks are stored at either 4° C. (0.1 M) or −20° C. (<0.1M). MCF-7 cells are passaged twice a week with growth medium [D-MEM/F-12medium containing 10% (v/v) heat-inactivated fetal bovine serum. 1%(v/v) Penicillin-Streptomycin, and 2 mM glutaMax-1]. The cells aremaintained in vented flasks at 37° C. inside a 5% CO₂/95% humidified airincubator. One day prior to treatment, the cells are plated with growthmedium at 25,000 cells/well into 96 well plates and incubated at 37° C.overnight.

The cells are infected for 2 hr at 37° C. with 50 μl/well of a 1:10dilution of adenovirus 5-ERE-tk-luciferase in experimental medium[phenol red-free D-MEM/F-12 medium containing 10% (v/v) heat-inactivedcharcoal-stripped fetal bovine serum, 1% (v/v) Penicillin-Streptomycin,2 mM glutaMax-1, 1 mM sodium pyruvate]. The wells are then washed oncewith 150 μl of experimental medium. Finally, the cells are treated for24 hr at 37° C. in replicates of 8 wells/treatment with 150 μl/well ofvehicle (≦0.1% v/v DMSO) or compound that is diluted ≧1000-fold intoexperimental medium.

Initial screening of test compounds is done at a single dose of 1 μMthat is tested alone (estrogen receptor agonist mode) or in combinationwith 0.1 nM 17β-estradiol (EC₈₀; estrogen receptor antagonist mode).Each 96 well plate also includes a vehicle control group (0.1% v/v DMSO)and an estrogen receptor agonist control group (either 0.1 or 1 nM17β-estradiol). Dose-response experiments are performed in either theestrogen receptor agonist and/or estrogen receptor antagonist modes onactive compounds in log increases from 10⁻¹⁴ to 10⁻⁵ M. From thesedose-response curves, EC₅₀ and IC₅₀ values, respectively, are generated.The final well in each treatment group contains 5 μl of 3×10⁻⁵ MICI-182,780 (10⁻⁶ M final concentration) as an estrogen receptorantagonist control.

After treatment, the cells are lysed on a shaker for 15 min with 25μl/well of 1× cell culture lysis reagent (Promega Corporation). The celllysates (20 μl) are transferred to a 96 well luminometer plate, andluciferase activity is measured in a MicroLumat LB 96 P luminometer (EG& G Berthold) using 100 μl/well of luciferase substrate (PromegaCorporation). Prior to the injection of substrate, a 1 second backgroundmeasurement is made for each well. Following the injection of substrate,luciferase activity is measured for 10 seconds after a 1 second delay.The data are transferred from the luminometer to a Macintosh personalcomputer and analyzed using the JMP software (SAS Institute); thisprogram subtracts the background reading from the luciferase measurementfor each well and then determines the mean and standard deviation ofeach treatment.

The luciferase data are transformed by logarithms, and the HuberM-estimator is used to down-weight the outlying transformedobservations. The JMP software is used to analyze the transformed andweighted data for one-way ANOVA (Dunnett's test). The compoundtreatments are compared to the vehicle control results in the estrogenreceptor agonist mode, or the positive estrogen receptor agonist controlresults (0.1 nM 17β-estradiol) in the estrogen receptor antagonist mode.For the initial single dose experiment, if the compound treatmentresults are significantly different from the appropriate control(p<0.05), then the results are reported as the percent relative to the17β-estradiol control [i.e., ((compound—vehicle control)/(17β-estradiolcontrol—vehicle control))×100)]. The JMP software is also used todetermine the EC₅₀ and/or IC₅₀ values from the non-linear dose-responsecurves.

Evaluation of Uterotrophic Activity

Uterotrophic activity of a test compound can be measured according tothe following standard pharmacological test procedures.

Procedure 1: Sexually immature (18 days of age) Sprague-Dawley rats areobtained from Taconic and provided unrestricted access to a casein-baseddiet (Purina Mills 5K96C) and water. On day 19, 20 and 21 the rats aredosed subcutaneously with 17α-ethinyl-17β-estradiol (0.06 μg/rat/day),test compound or vehicle (50% DMSO/50% Dulbecco's PBS). To assessestrogen receptor antagonist, compounds are coadministered with17α-ethinyl-17β-estradiol (0.06 μg/rat/day). There are six rats/groupand they are euthanized approximately 24 hours after the last injectionby CO₂ asphyxiation and pneumothorax. Uteri are removed and weighedafter trimming associated fat and expressing any internal fluid. Atissue sample can also be snap frozen for analysis of gene expression(e.g. complement factor 3 mRNA).

Procedure 2: Sexually immature (18 days of age) 129 SvE mice areobtained from Taconic and provided unrestricted access to a casein-baseddiet (Purina Mills 5K96C) and water. On day 22, 23, 24 and 25 the miceare dosed subcutaneously with compound or vehicle (corn oil). There aresix mice/group and they are euthanized approximately 6 hours after thelast injection by CO₂ asphyxiation and pneumothorax. Uteri are removedand weighed after trimming associated fat and expressing any internalfluid.

Evaluation of Osteoporosis and Lipid Modulation (Cardioprotection)

Female Sprague-Dawley rats, ovariectomized or sham operated, areobtained 1 day after surgery from Taconic Farms (weight range 240-275g). They are housed 3 or 4 rats/cage in a room on a 12/12 (light/dark)schedule and provided with food (Purina 5K96C rat chow) and water adlibitum. Treatment for all studies begin 1 day after arrival and ratsare dosed 7 days per week as indicated for 6 weeks. A group of agematched sham operated rats not receiving any treatment serve as anintact, estrogen replete control group for each study.

All test compounds are prepared in a vehicle of 50% DMSO (JT Baker,Phillipsburg, N.J.)/1× Dulbecco's phosphate saline (GibcoBRL, GrandIsland, N.Y.) at defined concentrations so that the treatment volume is0.1 mL/100 g body weight. 17β-estradiol is dissolved in corn oil (20μg/mL) and delivered subcutaneously, 0.1 mL/rat. All dosages areadjusted at three week intervals according to group mean body weightmeasurements, and given subcutaneously.

Five weeks after the initiation of treatment and one week prior to thetermination of the study, each rat is evaluated for bone mineral density(BMD). The total and trabecular density of the proximal tibia areevaluated in anesthetized rats using an XCT-960M (pQCT; StratecMedizintechnik, Pforzheim, Germany). The measurements are performed asfollows: Fifteen minutes prior to scanning, each rat is anesthetizedwith an intraperitoneal injection of 45 mg/kg ketamine, 8.5 mg/kgxylazine, and 1.5 mg/kg acepromazine.

The right hind limb is passed through a polycarbonate tube with adiameter of 25 mm and taped to an acrylic frame with the ankle joint ata 90° angle and the knee joint at 180°. The polycarbonate tube isaffixed to a sliding platform that maintains it perpendicular to theaperture of the pQCT. The platform is adjusted so that the distal end ofthe femur and the proximal end of the tibia is in the scanning field. Atwo dimensional scout view is run for a length of 10 mm and a lineresolution of 0.2 mm. After the scout view is displayed on the monitor,the proximal end of the tibia is located. The pQCT scan is initiated 3.4mm distal from this point. The pQCT scan is 1 mm thick, has a voxel(three dimensional pixel) size of 0.140 mm, and consists of 145projections through the slice.

After the pQCT scan is completed, the image is displayed on the monitor.A region of interest including the tibia but excluding the fibula isoutlined. The soft tissue is mathematically removed using an iterativealgorithm. The density of the remaining bone (total density) is reportedin mg/cm³. The outer 55% of the bone is mathematically peeled away in aconcentric spiral. The density of the remaining bone (Trabeculardensity) is reported in mg/cm³.

One week after BMD evaluation the rats are euthanized by CO₂asphyxiation and pneumothorax, and blood is collected for cholesteroldetermination. The uteri are also removed and the weighed after trimmingassociated fat and expressing any luminal fluid. Total cholesterol isdetermined using a Boehringer-Mannheim Hitachi 911 clinical analyzerusing the Cholesterol/HP kit. Statistics were compared using one-wayanalysis of variance with Dunnet's test.

Evaluation of Antioxidant Activity

Porcine aortas are obtained from an abattoir, washed, transported inchilled PBS, and aortic endothelial cells are harvested. To harvest thecells, the intercostal vessels of the aorta are tied off and one end ofthe aorta clamped. Fresh, sterile filtered, 0.2% collagenase (Sigma TypeI) is placed in the vessel and the other end of the vessel then clampedto form a closed system. The aorta is incubated at 37° C. for 15-20minutes, after which the collagenase solution is collected andcentrifuged for 5 minutes at 2000×g. Each pellet is suspended in 7 mL ofendothelial cell culture medium consisting of phenol red free DMEM/Ham'sF12 media supplemented with charcoal stripped FBS (5%), NuSerum (5%),L-glutamine (4 mM), penicillin-streptomycin (1000 U/ml, 100 μg/ml) andgentamycin (75 μg/ml), seeded in 100 mm petri dish and incubated at 37°C. in 5% CO₂. After 20 minutes, the cells are rinsed with PBS and freshmedium added, this was repeated again at 24 hours. The cells areconfluent after approximately 1 week. The endothelial cells areroutinely fed twice a week and, when confluent, trypsinized and seededat a 1:7 ratio. Cell mediated oxidation of 12.5 μg/mL LDL is allowed toproceed in the presence of the compound to be evaluated (5 μM) for 4hours at 37° C. Results are expressed as the percent inhibition of theoxidative process as measured by the TBARS (thiobarbituric acid reactivesubstances) method for analysis of free aldehydes [Yagi, BiochemicalMedicine 15: 212-6 (1976)].

Progesterone Receptor mRNA Regulation Standard Pharmacological TestProcedure

This test procedure can be used to evaluate the estrogenic orantiestrogenic activity of compounds from this invention [Shughrue, etal., Endocrinology 138: 5476-5484 (1997)].

Rat Hot Flush Test Procedure

The effect of test compounds on hot flushes can be evaluated in astandard pharmacological test procedure which measures the ability of atest compound to blunt the increase in tail skin temperature whichoccurs as morphine-addicted rats are acutely withdrawn from the drugusing naloxone [Merchenthaler, et al., Maturitas 30: 307-16 (1998)]. Itcan also be used to detect estrogen receptor antagonist activity byco-dosing test compound with the reference estrogen.

Evaluation of Vasomotor Function in Isolated Rat Aortic Rings

Sprague-Dawley rats (240-260 grams) are divided into 4 groups:

-   1. Normal non-ovariectomized (intact)-   2. Ovariectomized (ovex) vehicle treated-   3. Ovariectomized 17β-estradiol treated (1 mg/kg/day)-   4. Ovariectomized animals treated with test compound (various doses)

Animals are ovariectomized approximately 3 weeks prior to treatment.Each animal receives either 17-β estradiol sulfate (1 mg/kg/day) or testcompound suspended in distilled, deionized water with 1% tween-80 bygastric gavage. Vehicle treated animals received an appropriate volumeof the vehicle used in the drug treated groups.

Animals are euthanized by CO₂ inhalation and exsanguination. Thoracicaortae are rapidly removed and placed in 37° C. physiological solutionwith the following composition (mM): NaCl (54.7), KCl (5.0), NaHCO₃(25.0), MgCl₂ 2H₂O (2.5), D-glucose (11.8) and CaCl₂ (0.2) gassed withCO₂—O₂, 95% /5% for a final pH of 7.4. The advantitia is removed fromthe outer surface and the vessel is cut into 2-3 mm wide rings. Ringsare suspended in a 10 mL tissue bath with one end attached to the bottomof the bath and the other to a force transducer. A resting tension of 1gram is placed on the rings. Rings are equilibrated for 1 hour, signalsare acquired and analyzed.

After equilibration, the rings are exposed to increasing concentrationsof phenylephrine (10⁻⁸ to 10⁻⁴M) and the tension recorded. Baths arethen rinsed 3 times with fresh buffer. After washout, 200 mM L-NAME isadded to the tissue bath and equilibrated for 30 minutes. Thephenylephrine concentration response curve is then repeated.

Evaluation of Cardioprotective Activity

Apolipoprotein E-deficient C57/B1J (apo E KO) mice are obtained fromTaconic Farms. All animal procedures are performed under strictcompliance to IACUC guidelines. Ovariectomized female apo E KO mice, 4-7weeks of age, are housed in shoe-box cages and were allowed free accessto food and water. The animals are randomized by weight into groups(n=12-15 mice per group). The animals are dosed with test compounds orestrogen (17β-estradiol sulfate at 1 mg/kg/day) in the diet using aPrecise-dosing Protocol, where the amount of diet consumed is measuredweekly, and the dose adjusted accordingly, based on animal weight. Thediet used is a Western-style diet (57U5) that is prepared by Purina andcontains 0.50% cholesterol, 20% lard and 25 IU/KG Vitamin E. The animalsare dosed/fed using this paradigm for a period of 12 weeks. Controlanimals are fed the Western-style diet and receive no compound. At theend of the study period, the animals are euthanized and plasma samplesobtained. The hearts are perfused in situ, first with saline and thenwith neutral buffered 10% formalin solution.

For the determination of plasma lipids and lipoproteins, totalcholesterol and triglycerides are determined using enzymatic methodswith commercially available kits from Boehringer Mannheim and WakoBiochemicals, respectively and analyzed using the Boehringer MannheimHitachii 911 Analyzer. Separation and quantification of plasmalipoproteins were performed using FPLC size fractionation. Briefly,50-100 mL of serum is filtered and injected into Superose 12 andSuperose 6 columns connected in series and eluted at a constant flowrate with 1 mM sodium EDTA and 0.15 M NaCl. Areas of each curverepresenting VLDL. LDL and HDL are integrated using Waters Millennium™software, and each lipoprotein fraction is quantified by multiplying theTotal Cholesterol value by the relative percent area of each respectivechromatogram peak.

For the quantification of aortic atherosclerosis, the aortas arecarefully isolated and placed in formalin fixative for 48-72 hoursbefore handling. Atherosclerotic lesions are identified using Oil Red Ostaining. The vessels are briefly destained, and then imaged using aNikon SMU800 microscope fitted with a Sony 3CCD video camera system inconcert with IMAQ Configuration Utility (National Instrument) as theimage capturing software. The lesions are quantified en face along theaortic arch using a custom threshold utility software package (ColemanTechnologies). Automated lesion assessment is performed on the vesselsusing the threshold function of the program, specifically on the regioncontained within the aortic arch from the proximal edge of thebrachio-cephalic trunk to the distal edge of the left subclavian artery.Aortic atherosclerosis data are expressed as percent lesion involvementstrictly within this defined luminal area.

Evaluation of Cognition Enhancement

Ovariectomized rats (n=50) are habituated to an 8-arm radial arm mazefor 10-min periods on each of 5 consecutive days. Animals arewater-deprived prior to habituation and testing. A 100 μL aliquot ofwater placed at the ends of each arm serves as reinforcement.Acquisition of a win-shift task in the radial arm maze is accomplishedby allowing the animal to have access to one baited arm. After drinking,the animal exits the arm and re-enters the central compartment, where itnow has access to the previously visited arm or to a novel arm. Acorrect response is recorded when the animal chooses to enter a novelarm. Each animal is given 5 trials per day for 3 days. After the lastacquisition trial, the animals are assigned to one of the following 4groups:

-   -   1. Negative controls: injected with 10% DMSO/sesame oil vehicle        once daily for 6 days (1 mL/kg, SC)    -   2. Positive controls: injected with 17β-estradiol benzoate for 2        days and tested 4 days after the second injection (17β-estradiol        benzoate at 10 μg/0.1 mL per rat)    -   3. Estradiol: 17β-estradiol will be injected daily for 6 days        (20 μg/kg, SC)    -   4. Test compound: injected daily for 6 days (doses vary).        All injections will begin after testing on the last day of        acquisition. The last injection for groups 1, 3, and 4 will take        place 2 hours before testing for working memory.

The test for working memory is a delayed non-matching-to-sample task(DNMS) utilizing delays of 15, 30, or 60 seconds. This task is avariation of the acquisition task in which the rat is placed in thecentral arena and allowed to enter one arm as before. A second arm isopened once the rat traverses halfway down the first arm, and again therat is required to choose this arm. When it has traveled halfway downthis second arm, both doors are closed and the delay is instituted. Oncethe delay has expired, both of the original two doors, and a third noveldoor, are opened simultaneously. A correct response is recorded when theanimal travels halfway down the third, novel arm. An incorrect responseis recorded when the animal travels halfway down either the first orsecond arms. Each animal will receive 5 trials at each of the threedelay intervals for a total of 15 trials per subject.

Evaluation of Effect on Pleurisy

The ability to reduce the symptoms of experimentally-induced pleurisy inrats can be evaluated according to the procedure of Cuzzocrea[Endocrinology 141: 1455-63 (2000)].

Evaluation of Protection Against Glutamate-Induced Cytotoxicity(Neuroprotection)

The neuroprotective activity of compounds of this invention can beevaluated in an in vitro standard pharmacological test procedure usingglutamate challenge [Zaulyanov, et al., Cellular & MolecularNeurobiology 19: 705-18 (1999); Prokai, et al., Journal of MedicinalChemistry 44: 110-4 (2001)].

Evaluation in the Mammary End Bud Test Procedure

Estrogens are required for full ductal elongation and branching of themammary ducts, and the subsequent development of lobulo-alveolar endbuds under the influence of progesterone. In this test procedure, themammotrophic activity of selected compounds of the invention can beevaluated according to the following standard pharmacological testprocedure. Twenty-eight day old Sprague-Dawley rats (Taconic Farms,Germantown, N.Y.) are ovariectomized and rested for nine days. Animalsare housed under a 12-hour light/dark cycle, fed a casein-based PurinaLaboratory Rodent Diet 5K96 (Purina, Richmond, Ind.) and allowed freeaccess to water. Rats were then dosed subcutaneously for six days withvehicle (50% DMSO (JT Baker, Phillipsburg, N.J.)/50% 1× Dulbecco'sPhosphate buffered saline (GibcoBRL, Grand Island, N.Y.), 17β-estradiol(0.1 mg/kg) or test compound (20 mg/kg). For the final three days, ratsare also dosed subcutaneously with progesterone (30 mg/kg). On theseventh day, rats are euthanised and a mammary fat pad excised. This fatpad is analyzed for casein kinase II mRNA as a marker of end budproliferation. Casein kinase II mRNA is anlayzed by real-time RT-PCR.Briefly, RNA is isolated following Trizol (GibcoBRL, Grand Island, N.Y.)according to the manufacture's directions, Samples are treated withDNAse I using DNA-free kit (Ambion), and casein kinase II mRNA levelsare measured by real-time RT-PCR using the Taqman Gold procedure (PEApplied Biosystems). A total of 50 ng of RNA is analyzed in triplicateusing casein kinase II specific primer pair (5′ primer,CACACGGATGGCGCATACT (SEQ ID No: 1); 3′ primer, CTCGGGATGCACCATGAAG (SEQID No: 2)) and customized probe (TAMRA-CGGCACTGGTTTCCCTCACATGCT-FAM (SEQID No. 3)). Casein kinase II mRNA levels are normalized to 18s ribosomalRNA contained within each sample reaction using primers and probesupplied by PE Applied Biosystems.

Evaluation in the HLA Rat Standard Pharmacological Test Procedure forInflammatory Bowel Disease

Representative compounds can be evaluated in the HLA rat standardpharmacological test procedure which emulates inflammatory bowel diseasein humans. The following briefly describes the procedure used andresults obtained. Male HLA-B27 rats are obtained from Taconic andprovided unrestricted access to food (PMI Lab diet 5001) and water. Ratsare dosed subcutaneously once per day with either vehicle (50% DMSO/50%1× Dulbecco's Phosphate Buffered Saline) or test compound (0.1 to 10mg/kg) for at least one week. Stool quality is observed daily and gradedaccording to the following scale: Diarrhea=3; soft stool=2; normalstool=1. At the end of the study, serum is collected and stored at −70°C. A section of colon is prepared for histological analysis and anadditional segment is analyzed for myeloperoxidase activity.

For histological analysis, colonic tissue is immersed in 10% neutralbuffered formalin. Each specimen of colon is separated into four samplesfor evaluation. The formalin-fixed tissues are processed in a Tissue Tekvacuum infiltration processor (Miles, Inc; West Haven, Conn.) forparaffin embedding. The samples are sectioned at 5 μm and then stainedwith hematoxylin and eosin (H&E) for blinded histologic evaluationsusing a scale modified after Boughton-Smith. After the scores arecompleted the samples are unblinded, and data are tabulated and analyzedby ANOVA linear modeling with multiple mean comparisons. Sections ofcolonic tissue are evaluated for several disease indicators and givenrelative scores.

Evaluation in Three Models of Arthritis

Lewis rat assay of adjuvant-induced arthritis. Sixty, female, 12 weeksold, Lewis rats are housed according to standard facility operatingprocedures. They receive a standard regimen of food and water adlibitum. Each animal is identified by a cage card indicating the projectgroup and animal number. Each rat number is marked by indelible inkmarker on the tail. At least 10-21 days before study they areanesthetized and ovariectomized by standard aseptic surgical techniques.

Freund's Adjuvant-Complete (Sigma Immuno Chemicals, St. Louis, Mo.) isused to induce arthritis, each mL containing 1 mg Mycobacteriumtuberculosis heat killed and dried, 0.85 mL mineral oil and 0.15 mLmannide monooleate Lot No. 084H8800.

The following are examples of two test procedures. Inhibition testprocedure: Thirty rats are injected intradermally with 0.1 mL ofFreund's Adjuvant-Complete at the base of the tail. The animals arerandomized to four groups, each group containing six rats. Each day, thegroups receive vehicle (50% DMSO (J T Baker, Phillipsburg, N.J/)/1×Dulbecco's phosphate saline (GibcoBRL, Grand Island, N.Y.)) or testcompound (0.1-10 mg/kg, administered subcutaneously). All rats begintreatment on Day 1.

Treatment test procedure: Thirty rats are injected intradermally with0.1 mL of Freund's Adjuvant-Complete at the base of the tail. Theanimals are randomized to four groups, each group containing six rats.Each day, the groups receive vehicle (50% DMSO (JT Baker, Phillipsburg,N.J.)/1× Dulbecco's phosphate saline (GibcoBRL, Grand Island, N.Y.)) ortest compound (0.1-10 mg/kg, administered subcutaneously). All ratsbegin treatment on Day 8 after adjuvant injection.

Statistical analysis is performed using Abacus Concepts Super ANOVA.(Abacus Concepts, Inc., Berkeley, Calif.). All of the parameters ofinterest are subjected to Analysis of Variance with Duncan's newmultiple range post hoc testing between groups. Data are expressedthroughout as mean±standard deviation (SD), and differences are deemedsignificant if p<0.05.

The degree of arthritis severity is monitored daily in terms of thefollowing disease indices: Hindpaw erythema, hindpaw swelling,tenderness of the joints, and movements and posture. An integer scale of0 to 3 is used to quantify the level of erythema (0=normal paw, 1=milderythema, 2=moderate erythema, 3=severe erythema) and swelling (0=normalpaw, 1=mild swelling, 2=moderate swelling, 3=severe swelling of the hindpaw). The maximal score per day is 12.

At the end of the study the rats are euthanized with CO₂, hindlimbsremoved at necropsy and fixed in 10% buffered formalin, and the tarsaljoints decalcified and embedded in paraffin. Histologic sections arestained with Hematoxylin and Eosin or Saffranin O—Fast Green stain.

Slides are coded so that the examiner is blinded to the treatmentgroups. Synovial tissue from tarsal joints is evaluated based onsynovial hyperplasia, inflammatory cell infiltration, and pannusformation [Poole and Coombs, International Archives of Allergy & AppliedImmunology 54: 97-113 (1977)] as outlined below.

Category Grade 1. Synovial lining cells a. No change 0 b. Cellsenlarged, slightly thickened 1 c. Cells enlarged, increase in numbers, 2moderately thickened. No villus present d. Cells enlarged, thickened.Villlus present 3 2. Fibroplasia a. No change 0 b. Fibroplasia presentunder lining cells 1 c. Small areas of areolar tissue replaced 2 byfibrous tissue d. Replacement of areolar tissue by fibrous tissue 3 3.Inflammatory cells a. Occasionally seen. scattered throughout 0selection b. Cells present in small numbers in or just 1 under liningcell layer and/or around blood vessels. c. Small focal collection ofcells may be present 2 d. Large numbers of cells present in capsule 3and in or under lining cell layers. Large foci often seen. 4. Pannus a.Not detectable 0 b. Detectable 1

In addition, articular cartilage and bone is evaluated using Mankin'shistological grading system [Mankin, et al., Journal of Bone & JointSurgery—American Volume 53: 523-37 (1971)] as shown below.

Category Grade 1. Structure a. Normal 0 b. Surface irregularity 1 c.Pannus and surface irregularity 2 d. Clefts to transitional zone 3 e.Clefts to radial zone 4 f. Clefts to calcified zone 5 g. Completedisorganization 6 2. Cells a. Normal 0 b. Diffuse hypercellularity 1 c.Cloning 2 d. Hypocellularity 3 3. Safranin-O staining a. Normal 0 b.Slight reduction 1 c. Modest reduction 2 d. Severe reduction 3 e. No dyenoted 4 4. Tidemark integrity a. Intact 0 b. Crossed by blood vessels 1Evaluation in the HLA-B27 Rat Model of Arthritis

Representative compounds are evaluated in the HLA-B27 rat standardpharmacological test procedure which emulates arthritis in humans. Thefollowing briefly describes the procedure used. Male HLA-B27 rats areobtained from Taconic and provided unrestricted access to a food (PMILab diet 5001) and water. Rats are dosed subcutaneously once per daywith either vehicle (50% DMSO/50% 1× Dulbecco's Phosphate BufferedSaline) or test compound (0.1 to 10 mg/kg) for at least one week. Jointscores and histology are evaluated as described above for the Lewis ratmodel of adjuvant-induced arthritis.

Evaluation in the Collagen Induced Arthritis Models

Compounds are evaluated in BALB/c mice, 6-8 weeks of age, in whicharthritis is induced by monoclonal antibodies raised against type IIcollagen, plus lipopolysaccharide (LPS). The animals were administeredintravenously with a combination of 4 different mAbs totalling 4mg/mouse on day 0, and followed by intravenous 25 μg of LPS 72 hourslater (day 3). From day 3, one hour after LPS application, testedcompounds are give orally once daily for 15 days. For each animal,increase in volume of both hind paws is measured using a plethysmometerwith water cell (12 mm diameter) on days 0, 5, 7, 10, 14 and 17. Percentinhibition of increase in volume is calculated.

Evaluation in in vivo Models of Carcinogeneisis

The ability of compounds of this invention to treat and inhibit variousmalignancies or hyperprolific disorders can be evaluated in standardpharmacological test procedures that are readily available in theliterature, and include the following two procedures.

Breast cancer. Athymic nu/nu (nude) mice are obtained ovariectomizedfrom Charles River Laboratories (Wilmington, Mass.). One day prior totumor cell injection, animals are implanted with time-release pelletscontaining 0.36-1.7 mg 17β-estradiol (60 or 90 day release, InnovativeResearch of America, Sarasota, Fla.) or a placebo. The pellet isintroduced subcutaneously into the intrascapular region using a 10-gaugeprecision trochar. Subsequently, mice are injected subcutaneously intothe breast tissue with either 1×10⁷ MCF-7 cells or 1×10⁷ BG-1 cells. Thecells are mixed with an equal volume of matrigel, a basement membranematrix preparation to enhance tumor establishment. Test compounds can beevaluated either by dosing one day after tumor cell implantation(inhibition regimen) or after tumors have reached a certain size(treatment regimen). Compounds are administered either intraperitoneallyor orally in a vehicle of 1% tween-80 in saline each day. Tumor size isevaluated every three or seven days.

Colon cancer. The ability to treat or inhibit colon cancer can beevaluated in the test procedure of Smirnoff [Oncology Research 11:255-64 (1999)].

Evaluation of Neuroprotection in Two in vivo Test Procedures

Transient global ischemia in the Mongolian gerbil. The effect of testcompounds on preventing or treating brain injury in response to oxygendeprivation/reperfusion can be measured using the following testprocedure.

Female Mongolian gerbils (60-80 g; Charles River Laboratories, Kingston,N.Y.) are housed in the Wyeth-Ayerst animal care facility (AAALACcertified) with a 12-hour light, 12-hour dark photoperiod and freeaccess to tap water and a low-estrogen casein diet (Purina; Richmond,Ind.). After acclimation (3-5 days), gerbils are anesthetized withisoflurane (2-3% mixture with O₂), ovariectomized (Day 0). Beginning thefollowing morning (Day 1), gerbils are treated subcutaneously each daywith either vehicle (10% ETOH/com oil), 17β-estradiol (1 mg/kg) or anexperimental compound (0.1-20 mg/kg). On Day 6, gerbils (n=4-5/group)are anesthetized with isoflurane, the common carotid arteries visualizedvia a mid-line neck incision and both arteries simultaneously occludedfor 5 minutes with non-traumatic micro aneurysm clips. After occlusion,the clips are removed to allow cerebral reperfusion and the neckincision closed with wound clips. All animals are fasted overnight priorto the global ischemia surgery, a step that facilitates consistentischemic injury. On Day 12, gerbils are exposed to a lethal dose of CO₂,and the brains frozen on dry ice and stored at −80° C.

The degree of neuronal protection is evaluated by in situ hybridizationanalysis of neurogranin mRNA. Briefly, 20 μm coronal cryostat sectionsare collected on gelatin-coated slides, dried and stored at −80° C. Atthe time of processing, the desiccated slide boxes are warmed to roomtemperature, the slides postfixed in 4% paraformaldehyde, treated withacetic anhydride and then delipidated and dehydrated with chloroform andethanol. Processed section-mounted slides are then hybridized with 200μl (6×10⁶ DPM/slide) of an antisense or sense (control) riboprobe forNeurogranin (³⁵S-UTP-labeled NG-241; bases 99-340) in a 50% formamidehybridization mix and incubated overnight at 55° C. in a humidifiedslide chamber without coverslipping. The following morning, the slidesare collected in racks, immersed in 2×SSC (0.3 M NaCl, 0.03 M sodiumcitrate; pH 7.0)/10 mM DTT, treated with RNase A (20 μg/ml) and washed(2×30 min) at 67° C. in 0.1×SSC to remove nonspecific label. Afterdehydration, the slides are opposed to BioMax (BMR-1: Kodak) X-ray filmovernight.

The level of neurogranin hybridization signal is used to quantitativelyassess the degree of neuronal loss in the CA1 region after injury and toevaluate the efficacy of 17β-estradiol and experimental compounds.Neurogranin mRNA is selected for these studies because it is highlyexpressed in the hippocampal neurons including CA1, but absent in gliaand other cell types present in this brain region. Therefore,measurement of the amount of neurogranin mRNA present representssurviving neurons. Relative optical density measurements of neurograninhybridization signal are obtained from film autoradiograms with acomputer based image analysis system (C-Imaging Inc., Pittsburgh, Pa.).The results from 6 sections (40 μm apart) per animal are averaged andstatistically evaluated. Numerical values are reported as the mean±SEM.One-way analysis of variance is used to test for differences in thelevel of neurogranin mRNA and all statements of non-difference in theresults section imply that p>0.05.

Middle cerebral artery occlusion in mice. Neuroprotection can beevaluated according to the test procedures described by Dubal [see,Dubal, et al., Proceedings of the National Academy of Sciences of theUnited States of America 98: 1952-1957 (2001), Dubal, et al., Journal ofNeuroscience 19: 6385-6393 (1999)].

Ovulation Inhibition Standard Pharmacological Test Procedure

The test procedure is used to determine whether test compounds caninhibit or change the timing of ovulation. It can also be used todetermine the number of oocytes ovulated [Lundeen, et al., J SteroidBiochem Mol Biol 78: 137-143 (2001)].

Transplantation Rejection

To test the ability of the test compounds to prevent transplantrejection. Compounds can be tested in animal models of hearttransplantation (Stetson et al. Circulation 104:676-682 (2001) ortransplant atherosclerosis (Deitrich et al. Arterioscler. Thromb. VascBiol. 20:343-352 (2000). Lou et al., Circulation 94:3355-3361 (1996).

Prevention of Restenosis

The test procedure is used to determine whether test compounds caninhibit vascular smooth muscle cell proliferation after carotid arteryinjury similar to what occurs after balloon angioplasty. The testcompounds can be tested in animal models previously described (Karas etal. Circ Res. 89:534-539 (2001), Cerek et al. Atherosclerosis 131:59-66(1997).

Treatment of Myocardial Infarction

Test compounds can be tested in animal models of ischemia/reperfusion todetermine whether they would inhibit cell death occurring during amyocardial infarction. The compounds can be tested in models describedpreviously (Delyani et al. J Mol & Cell Cardiology 28:1001-1008 (1996),Izumi et al. J Clin Invest. 108:203-213 (2001) & Chandrasekar et al.Circulation 103:2296-2302 (2001)).

Treatment for Myocarditis and Congestive Heart Failure

Test compounds can be tested in models of heart failure to determinewhether compounds could be an effective therapy and improve cardiacfunction. Compounds can be tested in animals as described previously(Yokoseki et al. Circ Res. 89:1-9 (2001), Wallen et al. Hypertension36:774-779 (2000) & Toshiaki et al. Circulation 104:1094-1103 (2001)).

Treatment for Diabetes

Test compounds can be tested in models of diabetes to determine theireffect on reversal of obesity and diet-induced insulin resistance.Compounds can be tested in animal models as previously described (Yuanet al. Science 293:1673-1677 (2001).

Treatment for Asthma

Pulmonary Inflammation Model Sensitize mice with OVA emulsified in alumon days 0 and 14 (ip injection). On days 28 and 29 challenge with anaerosol of OVA for 20 min (1%-5% OVA) and then on Day 30 the animals aresacrificed and harvest BAL and/or lung tissue for analysis of pulmonan,inflammation.

Airway Hyperresponsiveness. This model is similar to that describedabove however animals are challenged on 3 consecutive days with anaerosol of OVA and airway hyperresponsiveness is measured 48 h after thelast challenge BAL can also be taken at this stage if required.

To look more directly at the effects of mast cells in conjunction withER, may use a passive cutaneous anaphylaxis model in which IgE isinjected into the ear and then 24 hours later inject DNP-HSA iv. Measureear thickness and an early and late phase reaction. Furthermore, fixtissues in K2 and embed in Epoxy resin and cut lum sections. These canbe stained for mast cells and quantitiate the degree of mast celldegranulation.

Based on the results obtained in the standard pharmacological testprocedures, the compounds of this invention are selectiveanti-inflammatory compounds described herein useful for the treatmentand prevention of chronic inflammatory diseases without stimulatinguterine and breast cell proliferation as found with classic estrogens.

Accordingly, the compounds of this invention are useful in treating orinhibiting osteoporosis and in the inhibition of bone demineralizationwhich may result from an imbalance in a individual's formation of newbone tissues and the resorption of older tissues, leading to a net lossof bone. Such bone depletion results in a range of individuals,particularly in post-menopausal women, women who have undergonebilateral oophorectomy, those receiving or who have received extendedcorticosteroid therapies, those experiencing gonadal dysgenesis, andthose suffering from Cushing's syndrome. Special needs for bone,including teeth and oral bone, replacement can also be addressed usingthese compounds in individuals with bone fractures, defective bonestructures, and those receiving bone-related surgeries and/or theimplantation of prosthesis. In addition to those problems describedabove, these compounds can be used in treatment or inhibition forosteoarthritis, hypocalcemia, hypercalcemia, Paget's disease,osteomalacia, osteohalisteresis, multiple myeloma and other forms ofcancer having deleterious effects on bone tissues.

The compounds of this invention are also active in the brain and aretherefore useful for inhibiting or treating Alzheimer's disease,cognitive decline, decreased libido, senile dementia, neurodegenerativedisorders, depression, anxiety, insomnia, schizophrenia, andinfertility. The compounds of this invention are also useful in treatingor inhibiting benign or malignant abnormal tissue growth includingglomerulosclerosis, prostatic hypertrophy, uterine leiomyomas, breastcancer, scleroderma, fibromatosis, endometriosis, endometrial cancer,polycystic ovary syndrome, endometrial polyps, benign breast disease,adenomyosis, ovarian cancer, melanoma, prostate cancer, cancers of thecolon, CNS cancers, such as glioma or astioblastomia.

The compounds of this invention are cardioprotective and areantioxidants, and are useful in lowering cholesterol, triglycerides,Lp(a), and LDL levels; inhibiting or treating hypercholesteremia,hyperlipidemia, cardiovascular disease, atherosclerosis, peripheralvascular disease, restenosis, and vasospasm, and inhibiting vascularwall damage from cellular events leading toward immune mediated vasculardamage.

The compounds of this invention are also useful in treating disordersassociated with inflammation or autoimmune diseases, includinginflammatory bowel disease (Crohn's disease, ulcerative colitis,indeterminate colitis), arthritis (rheumatoid arthritis,spondyloarthropathies, osteoarthritis), pleurisy, ischemia/reperfusioninjury (e.g. stroke, transplant rejection, myocardial infarction, etc.),asthma, giant cell arteritis, prostatitis, uveitis, psoriasis, multiplesclerosis, systemic lupus erythematosus and sepsis.

The compounds of this invention are also useful in treating orinhibiting ocular disorders including cataracts, uveitis, and maculardegeneration and in treating skin conditions such as aging, alopecia,and acne.

The compounds of this invention are also useful in treating orinhibiting metabolic disorders such as type-Hi diabetes, of lipidmetabolism, appetite (e.g. anorexia nervosa and bulimia).

Compounds in this invention are also useful in treating or inhibitingbleeding disorders such as hereditary hemorrhagic telangiectasia,dysfunctional uterine bleeding, and combating hemorrhagic shock.

The compounds of this invention are useful in disease states whereamenorrhea is advantageous, such as leukemia, endometrial ablations,chronic renal or hepatic disease or coagulation diseases or disorders.

All patents, publications, and other documents cited herein are herebyincorporated by reference in their entirety.

1. A compound of the formula:

or pharmaceutically acceptable salt thereof, wherein: R₁ is hydrogen,alkyl, halogen, aryl, thienyl, furanyl, or pyrrolyl; R₂, R₃, R₅, and R₆are, independently, hydrogen, hydroxy, lower alkyl, alkoxy, or halogen;R₄ is alkyl; R₇ is hydrogen, methyl, —(C═O)R₁₆, —S(O)₂R₁₇, or—S(O)₂N(R₁₈)(R₁₉); R₁₆ is alkyl; R₁₇ is alkyl, alkenyl or alkynyl; andR₁₈ and R₁₉ are, independently, hydrogen, alkyl, alkenyl, or alkynyl. 2.The compound of claim 1 or pharmaceutically acceptable salt thereofwherein R₅ and R₆ are H.
 3. The compound of claim 1 having the formula:

or pharmaceutically acceptable salt thereof.
 4. The compound of claim 1or pharmaceutically acceptable salt thereof wherein R₁ is methyl, ethyl,halogen, phenyl, or thienyl.
 5. The compound of claim 4 orpharmaceutically acceptable salt thereof wherein R₁ is methyl, ethyl,bromo, phenyl, or thienyl.
 6. The compound of claim 1 orpharmaceutically acceptable salt thereof wherein R₂ is H or Br.
 7. Thecompound of claim 1 or pharmaceutically acceptable salt thereof whereinR₃ is H or Br.
 8. The compound of claim 1 or pharmaceutically acceptablesalt thereof wherein R₂ and R₃ are each, independently, H or Br.
 9. Thecompound of claim 1 or pharmaceutically acceptable salt thereof whereinR₄ is methyl or ethyl.
 10. The compound of claim 1 or pharmaceuticallyacceptable salt thereof wherein R₇ is H or methyl.
 11. A compound ofthat is: 4-[(5-Methylthieno[3,2-c]isoquinolin-4(5H)-yl)sulfonyl]phenol,4-[(5-Ethylthieno[3,2-c]isoquinolin-4(5H)-yl)sulfonyl]phenol,4-[(2-Bromo-5-methylthieno[3,2-c]isoquinolin-4(5H) yl)sulfonyl]phenol,4-{[(5R)-2-bromo-5-methylthieno[3,2-c]isoquinolin-4-(5H)-yl]sulfonyl}phenol,4-{[(5S)-2-bromo-5-methylthieno[3,2-c]isoquinolin-4-(5H)-yl]sulfonyl}phenol,4-[(2-Bromo-5-ethylthieno[3,2-c]isoquinolin-4(5H)-yl)sulfonyl]phenol,4-[(5-Ethyl-2-thien-3-ylthieno[3,2-c]isoquinolin-4(5H)-yl)sulfonyl]phenol,4-[(5-Ethyl-2-phenylthieno[3,2-c]isoquinolin-4(5H)-yl)sulfonyl]phenol,2-Bromo-4-[(2-bromo-5-methylthieno[3,2-c]isoquinolin-4(5H)-yl)sulfonyl]phenol,2,6-Dibromo-4-[(2-bromo-5-methylthieno[3,2-c]isoquinolin-4(5H)-yl)sulfonyl]phenol,or 4-[(2,5-Diethylthieno[3,2-c]isoquinolin-4(5H)-yl )sulfonyl]phenol, orpharmaceutically acceptable salt thereof.
 12. A pharmaceuticalcomposition comprising a compound of claim 1 or pharmaceuticallyacceptable salt thereof and a pharmaceutical carrier.